Activation of HCV-specific T cells
The invention provides a method of activating hepatitis C virus (HCV)-specific T cells, including CD4+ and CD8+ T cells. HCV-specific T cells are activated using fusion proteins comprising HCV NS3, NS4, NS5a, and NS5b polypeptides, polynucleotides encoding such fusion proteins, or polypeptide or polynucleotide compositions containing the individual components of these fusions. The method can be used in model systems to develop HCV-specific immunogenic compositions, as well as to immunize a mammal against HCV.
This application is a continuation of U.S. application Ser. No. 10/281,341, filed Oct. 25, 2002, which is a continuation-in-part of U.S. application Ser. No. 09/698,874, filed Oct. 27, 2000, from which applications priority is claimed under 35 USC §120. U.S. application Ser. No. 09/698,874 claims the benefit of provisional patent application Ser. No. 60/161,713, filed Oct. 27, 1999 under 35 USC §119(e)(1). The foregoing applications are incorporated herein by reference in their entireties.
TECHNICAL AREA OF THE INVENTIONThe invention relates to the activation of hepatitis C virus(HCV)-specific T cells. More particularly, the invention relates to the use of multiple HCV polypeptides, either alone or as fusions, to stimulate cell-mediated immune responses, such as to activate HCV-specific T cells.
BACKGROUND OF THE INVENTIONHepatitis C virus (HCV) infection is an important health problem with approximately 1% of the world's population infected with the virus. Over 75% of acutely infected individuals eventually progress to a chronic carrier state that can result in cirrhosis, liver failure, and hepatocellular carcinoma. See Alter et al. (1992) N. Engl. J. Med. 327:1899-1905; Resnick and Koff. (1993) Arch. Intem. Med. 153:1672-1677; Seeff (1995) Gastrointest. Dis. 6:20-27; Tong et al. (1995) N. Engl. J. Med. 332:1463-1466.
Despite extensive advances in the development of pharmaceuticals against certain viruses like HIV, control of acute and chronic HCV infection has had limited success (Hoofnagle and di Bisceglie (1997) N. Engl. J. Med. 336:347-356). In particular, generation of a strong cytotoxic T lymphocyte (CTL) response is thought to be important for the control and eradication of HCV infections. Thus, there is a need in the art for effective methods of inducing strong CTL responses against HCV.
SUMMARY OF THE INVENTIONIt is an object of the invention to provide reagents and methods for stimulating immune responses, such as activating T cells which recognize epitopes of HCV polypeptides. This and other objects of the invention are provided by one or more of the embodiments described below.
The invention provides HCV proteins useful for stimulating immune responses, such as activating HCV-specific T cells. One embodiment provides a fusion protein that comprises HCV polypeptides, wherein the HCV polypeptides consist essentially of an NS3, an NS4, an NS5a polypeptide, and optionally a core polypeptide. In certain embodiments, the fusion protein includes an NS5b polypeptide.
In certain embodiments, at least one of the HCV polypeptides is derived from a different strain of HCV than the other polypeptides.
The invention also provides compositions comprising any of these fusion proteins and a pharmaceutically acceptable excipient. In certain embodiments, the compositions further comprise an adjuvant, a CpG polynucleotide and/or the fusion protein is adsorbed to or entrapped within a microparticle or ISCOM. The compositions can further comprise a polynucleotide encoding an E1E2 complex. The E1E2 polynucleotide can also be adsorbed to or entrapped withing a microparticle.
Another embodiment provides a composition comprising HCV polypeptides and a pharmaceutically acceptable excipient. The HCV polypeptides consist essentially of an NS3, an NS4, an NS5a polypeptide, and optionally a core polypeptide. In certain embodiments, the composition includes an NS5b polypeptide. In other embodiments, the compositions further comprise an adjuvant, a CpG polynucleotide and/or one or more of the HCV polypeptides is adsorbed to or entrapped within a microparticle or ISCOM. The compositions can further comprise a polynucleotide encoding an E1E2 complex. The E1E2 polynucleotide can also be adsorbed to or entrapped withing a microparticle. Moreover, one of the HCV polypeptides may be derived from a different strain of HCV than the others.
Even another embodiment of the invention provides an isolated and purified polynucleotide which encodes a fusion protein as described above. In additional embodiments, the fusion proteins further include a polynucleotide encoding an E1E2 complex.
Yet another embodiment of the invention provides a composition comprising the polynucleotides described above and a pharmaceutically acceptable excipient. In certain embodiments, the compositions further comprise an adjuvant and/or the polynucleotide may be adsorbed to or entrapped within a microparticle. The compositions can further comprise a polynucleotide encoding an E1E2 complex. The E1E2 polynucleotide can also be adsorbed to or entrapped withing a microparticle.
In a further embodiment, the invention provides a composition comprising HCV polynucleotides and a pharmaceutically acceptable excipient, wherein the HCV polynucleotides consist essentially of polynucleotides encoding an NS3, an NS4, an NS5a polypeptide, and optionally a core polypeptide. In certain embodiments, the composition also includes a polynucleotide encoding an NS5b polypeptide. The compositions may further comprise an adjuvant and/or one or more of the polynucleotides may be adsorbed to or entrapped within a microparticle. The compositions can further comprise a polynucleotide encoding an E1E2 complex. The E1E2 polynucleotide can also be adsorbed to or entrapped withing a microparticle. Additionally, one or more of the polynucleotides may be derived from a different strain of HCV than the others.
In another embodiment, the invention provides a method of activating T cells which recognize an epitope of an HCV polypeptide. T cells are contacted with any of the fusions, polynucleotides or compositions described above. A population of activated T cells recognizes an epitope of the NS3, NS4, NS5a, NS5b, core and/or E1E2 polypeptide.
In the proteins and polynucleotides above, the regions in the fusions need not be in the order in which they naturally occur in the native HCV polyprotein. Thus, for example, the NS5b polypeptide, if present, may be at the N- and/or C-terminus of the fusion, or may be located internally. Similarly, the E1 polypeptide may precede or follow the E2 polypeptide. The E1E2 polypeptide may also be part of the nonstructural fusion protein or may be provided separately, as an E1E2 complex, or as individual polypeptides.
Moreover, the NS3 polypeptide may include a modification to inhibit protease activity, such that cleavage of the fusion is inhibited. Such modifications are described more fully below. Additionally, the compositions can comprise more than one HCV nonstructural fusion protein, such as a fusion protein with NS3, NS4 and NS5a, and a fusion protein with NS3, NS4, NS5a, NS5b and E1E2. The E1E2 complexes, whether present separately or as part of the fusion, can have varying E1E2 polypeptides (described more fully below).
In certain embodiments, the nonstructural fusion protein consists of, from the amino terminus to the carboxyl terminus, an NS3, an NS4, an NS5a and, optionally, an NS5b polypeptide and the E1E2 complex consists of, from amino terminus to the carboxyl terminus, an E1 polypeptide and an E2 polypeptide.
The various polypeptides (and polynucleotides encoding therefor) are derived from the same HCV isolate, or from different strains and isolates including isolates having any of the various HCV genotypes, to provide increased protection against a broad range of HCV genotypes.
Yet another embodiment of the invention provides a method of stimulating an immune response, such as a cellular immune response, in a vertebrate subject by administering a composition as described herein. In certain embodiments, the composition activates T cells which recognize an epitope of an HCV polypeptide. T cells are contacted with a composition as described above. A population of activated T cells recognizes an epitope of one or more of the HCV polypeptide(s).
The invention thus provides methods and reagents for stimulating immune responses to HCV, such as for activating T cells which recognize epitopes of HCV polypeptides. These methods and reagents are particularly advantageous for identifying epitopes of HCV polypeptides associated with a strong CTL response and for immunizing mammals, including humans, against HCV.
The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, recombinant DNA techniques and immunology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); DNA Cloning, Vols. I and II (D. N. Glover ed.); Oligonucleotide Synthesis M. J. Gait ed.); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds.); Animal Cell Culture (R. K. Freshney ed.); Perbal, B., A Practical Guide to Molecular Cloning.
All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.
It must be noted that, as used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “an antigen” includes a mixture of two or more antigens, and the like.
The following amino acid abbreviations are used throughout the text:
-
- Alanine: Ala (A) Arginine: Arg (R)
- Asparagine: Asn (N) Aspartic acid: Asp (D)
- Cysteine: Cys (C) Glutamine: Gln (Q)
- Glutamic acid: Glu (E) Glycine: Gly (G)
- Histidine: His (H) Isoleucine: Ile (I)
- Leucine: Leu (L) Lysine: Lys (K)
- Methionine: Met (M) Phenylalanine: Phe (F)
- Proline: Pro (P) Serine: Ser (S)
- Threonine: Thr (T) Tryptophan: Trp (W)
- Tyrosine: Tyr (Y) Valine: Val (V)
In describing the present invention, the following terms will be employed, and are intended to be defined as indicated below.
The terms “polypeptide” and “protein” refer to a polymer of amino acid residues and are not limited to a minimum length of the product. Thus, peptides, oligopeptides, dimers, multimers, and the like, are included within the definition. Both full-length proteins and fragments thereof are encompassed by the definition. The terms also include postexpression modifications of the polypeptide, for example, glycosylation, acetylation, phosphorylation and the like. Furthermore, for purposes of the present invention, a “polypeptide” refers to a protein which includes modifications, such as deletions, additions and substitutions (generally conservative in nature), to the native sequence, so long as the protein maintains the desired activity. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification.
An HCV polypeptide is a polypeptide, as defined above, derived from the HCV polyprotein. The polypeptide need not be physically derived from HCV, but may be synthetically or recombinantly produced. Moreover, the polypeptide may be derived from any of the various HCV strains and isolates including isolates having any of the 6 genotypes of HCV described in Simmonds et al., J. Gen. Virol. (1993) 74:2391-2399 (e.g., strains 1, 2, 3, 4 etc.), as well as newly identified isolates, and subtypes of these isolates, such as HCV1a, HCV1b, etc. A number of conserved and variable regions are known between these strains and, in general, the amino acid sequences of epitopes derived from these regions will have a high degree of sequence homology, e.g., amino acid sequence homology of more than 30%, preferably more than 40%, when the two sequences are aligned. Thus, for example, the term “NS4” polypeptide refers to native NS4 from any of the various HCV strains, as well as NS4 analogs, muteins and immunogenic fragments, as defined further below.
By an “E1 polypeptide” is meant a molecule derived from an HCV E1 region. The mature E1 region of HCV-1 begins at approximately amino acid 192 of the polyprotein and continues to approximately amino acid 383, numbered relative to the full-length HCV-1 polyprotein. (See, FIGS. 1 and 3A-3C. Amino acids 192-383 of
By an “E2 polypeptide” is meant a molecule derived from an HCV E2 region. The mature E2 region of HCV-1 begins at approximately amino acid 383-385, numbered relative to the full-length HCV-1 polyprotein. (See, FIGS. 1 and 3A-3C. Amino acids 383-385 of
Representative E1 and E2 regions from HCV-1 are shown in
Furthermore, an “E1 polypeptide” or an “E2 polypeptide” as defined herein is not limited to a polypeptide having the exact sequence depicted in the Figures. Indeed, the HCV genome is in a state of constant flux in vivo and contains several variable domains which exhibit relatively high degrees of variability between isolates. A number of conserved and variable regions are known between these strains and, in general, the amino acid sequences of epitopes derived from these regions will have a high degree of sequence homology, e.g., amino acid sequence homology of more than 30%, preferably more than 40%, more than 60%, and even more than 80-90% homology, when the two sequences are aligned. It is readily apparent that the terms encompass E1 and E2 polypeptides from any of the various HCV strains and isolates including isolates having any of the 6 genotypes of HCV described in Simmonds et al., J. Gen. Virol. (1993) 74:2391-2399 (e.g., strains 1, 2, 3, 4 etc.), as well as newly identified isolates, and subtypes of these isolates, such as HCV1a, HCV1b etc.
Thus, for example, the term “E1” or “E2” polypeptide refers to native E1 or E2 sequences from any of the various HCV strains, as well as analogs, muteins and immunogenic fragments, as defined further below. The complete genotypes of many of these strains are known. See, e.g., U.S. Pat. No. 6,150,087 and GenBank Accession Nos. AJ238800 and AJ238799.
Additionally, the terms “E1 polypeptide” and “E2 polypeptide” encompass proteins which include modifications to the native sequence, such as internal deletions, additions and substitutions (generally conservative in nature). These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through naturally occurring mutational events. All of these modifications are encompassed in the present invention so long as the modified E1 and E2 polypeptides function for their intended purpose. Thus, for example, if the E1 and/or E2 polypeptides are to be used in vaccine compositions, the modifications must be such that immunological activity (i.e., the ability to elicit a humoral or cellular immune response to the polypeptide) is not lost.
By “E1E2” complex is meant a protein containing at least one E1 polypeptide and at least one E2 polypeptide, as described above. Such a complex may also include all or a portion of the p7 region which occurs immediately adjacent to the C-terminus of E2. As shown in FIGS. 1 and 3A-3C, the p7 region is found at positions 747-809, numbered relative to the full-length HCV-1 polyprotein (amino acid positions 575-637 of SEQ ID NO:4). A representative E1E2 complex which includes the p7 protein is termed “E1E2809” herein.
The mode of association of E1 and E2 in an E1E2 complex is immaterial. The E1 and E2 polypeptides may be associated through non-covalent interactions such as through electrostatic forces, or by covalent bonds. For example, the E1E2 polypeptides of the present invention may be in the form of a fusion protein which includes an immunogenic E1 polypeptide and an immunogenic E2 polypeptide, as defined above. The fusion may be expressed from a polynucleotide encoding an E1E2 chimera. Alternatively, E1E2 complexes may form spontaneously simply by mixing E1 and E2 proteins which have been produced individually. Similarly, when co-expressed and secreted into media, the E1 and E2 proteins can form a complex spontaneously. Thus, the term encompasses E1E2 complexes (also called aggregates) that spontaneously form upon purification of E1 and/or E2. Such aggregates may include one or more E1 monomers in association with one or more E2 monomers. The number of E1 and E2 monomers present need not be equal so long as at least one E1 monomer and one E2 monomer are present. Detection of the presence of an E1E2 complex is readily determined using standard protein detection techniques such as polyacrylamide gel electrophoresis and immunological techniques such as immunoprecipitation.
The terms “analog” and “mutein” refer to biologically active derivatives of the reference molecule, or fragments of such derivatives, that retain desired activity, such as the ability to stimulate a cell-mediated immune response, as defined below. In general, the term “analog” refers to compounds having a native polypeptide sequence and structure with one or more amino acid additions, substitutions (generally conservative in nature) and/or deletions, relative to the native molecule, so long as the modifications do not destroy immunogenic activity. The term “mutein” refers to peptides having one or more peptide mimics (“peptoids”), such as those described in International Publication No. WO 91/04282. Preferably, the analog or mutein has at least the same immunoactivity as the native molecule. Methods for making polypeptide analogs and muteins are known in the art and are described further below.
As explained above, analogs generally include substitutions that are conservative in nature, i.e., those substitutions that take place within a family of amino acids that are related in their side chains. Specifically, amino acids are generally divided into four families: (1) acidic—aspartate and glutamate; (2) basic—lysine, arginine, histidine; (3) non-polar—alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar—glycine, asparagine, glutamine, cysteine, serine threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids. For example, it is reasonably predictable that an isolated replacement of leucine with isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar conservative replacement of an amino acid with a structurally related amino acid, will not have a major effect on the biological activity. For example, the polypeptide of interest may include up to about 5-10 conservative or non-conservative amino acid substitutions, or even up to about 15-25 conservative or non-conservative amino acid substitutions, or any integer between 5-25, so long as the desired function of the molecule remains intact. One of skill in the art may readily determine regions of the molecule of interest that can tolerate change by reference to Hopp/Woods and Kyte-Doolittle plots, well known in the art.
By “modified NS3” is meant an NS3 polypeptide with a modification such that protease activity of the NS3 polypeptide is disrupted. The modification can include one or more amino acid additions, substitutions (generally non-conservative in nature) and/or deletions, relative to the native molecule, wherein the protease activity of the NS3 polypeptide is disrupted. Methods of measuring protease activity are discussed further below.
By “fragment” is intended a polypeptide consisting of only a part of the intact full-length polypeptide sequence and structure. The fragment can include a C-terminal deletion and/or an N-terminal deletion of the native polypeptide. An “immunogenic fragment” of a particular HCV protein will generally include at least about 5-10 contiguous amino acid residues of the full-length molecule, preferably at least about 15-25 contiguous amino acid residues of the full-length molecule, and most preferably at least about 20-50 or more contiguous amino acid residues of the full-length molecule, that define an epitope, or any integer between 5 amino acids and the full-length sequence, provided that the fragment in question retains immunogenic activity, as measured by the assays described herein.
The term “epitope” as used herein refers to a sequence of at least about 3 to 5, preferably about 5 to 10 or 15, and not more than about 1,000 amino acids (or any integer therebetween), which define a sequence that by itself or as part of a larger sequence, binds to an antibody generated in response to such sequence. There is no critical upper limit to the length of the fragment, which may comprise nearly the full-length of the protein sequence, or even a fusion protein comprising two or more epitopes from the HCV polyprotein. An epitope for use in the subject invention is not limited to a polypeptide having the exact sequence of the portion of the parent protein from which it is derived. Indeed, viral genomes are in a state of constant flux and contain several variable domains which exhibit relatively high degrees of variability between isolates. Thus the term “epitope” encompasses sequences identical to the native sequence, as well as modifications to the native sequence, such as deletions, additions and substitutions (generally conservative in nature).
Regions of a given polypeptide that include an epitope can be identified using any number of epitope mapping techniques, well known in the art. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66 (Glenn E. Morris, Ed., 1996) Humana Press, Totowa, N.J. For example, linear epitopes may be determined by e.g., concurrently synthesizing large numbers of peptides on solid supports, the peptides corresponding to portions of the protein molecule, and reacting the peptides with antibodies while the peptides are still attached to the supports. Such techniques are known in the art and described in, e.g., U.S. Pat. No. 4,708,871; Geysen et al. (1984) Proc. Natl. Acad. Sci. USA 81:3998-4002; Geysen et al. (1986) Molec. Immunol. 23:709-715, all incorporated herein by reference in their entireties. Similarly, conformational epitopes are readily identified by determining spatial conformation of amino acids such as by, e.g., x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols, supra. Antigenic regions of proteins can also be identified using standard antigenicity and hydropathy plots, such as those calculated using, e.g., the Omiga version 1.0 software program available from the Oxford Molecular Group. This computer program employs the Hopp/Woods method, Hopp et al., Proc. Natl. Acad. Sci. USA (1981) 78:3824-3828 for determining antigenicity profiles, and the Kyte-Doolittle technique, Kyte et al., J. Mol. Biol. (1982) 157:105-132 for hydropathy plots.
For a description of various HCV epitopes, see, e.g., Chien et al., Proc. Natl. Acad. Sci. USA (1992) 89:10011-10015; Chien et al., J. Gastroent. Hepatol. (1993) 8:S33-39; Chien et al., International Publication No. WO 93/00365; Chien, D. Y., International Publication No. WO 94/01778; and U.S. Pat. Nos. 6,280,927 and 6,150,087, incorporated herein by reference in their entireties.
As used herein, the term “conformational epitope” refers to a portion of a full-length protein, or an analog or mutein thereof, having structural features native to the amino acid sequence encoding the epitope within the full-length natural protein. Native structural features include, but are not limited to, glycosylation and three dimensional structure. Preferably, a conformational epitope is produced recombinantly and is expressed in a cell from which it is extractable under conditions which preserve its desired structural features, e.g. without denaturation of the epitope. Such cells include bacteria, yeast, insect, and mammalian cells. Expression and isolation of recombinant conformational epitopes from the HCV polyprotein are described in e.g., International Publication Nos. WO 96/04301, WO 94/01778, WO 95/33053, WO 92/08734, which applications are herein incorporated by reference in their entirety.
As used herein the term “T-cell epitope” refers to a feature of a peptide structure which is capable of inducing T-cell immunity towards the peptide structure or an associated hapten. T-cell epitopes generally comprise linear peptide determinants that assume extended conformations within the peptide-binding cleft of MHC molecules, (Unanue et al., Science (1987) 236:551-557). Conversion of polypeptides to MHC class II-associated linear peptide determinants (generally between 5-14 amino acids in length) is termed “antigen processing” which is carried out by antigen presenting cells (APCs). More particularly, a T-cell epitope is defined by local features of a short peptide structure, such as primary amino acid sequence properties involving charge and hydrophobicity, and certain types of secondary structure, such as helicity, that do not depend on the folding of the entire polypeptide. Further, it is believed that short peptides capable of recognition by helper T-cells are generally amphipathic structures comprising a hydrophobic side (for interaction with the MHC molecule) and a hydrophilic side (for interacting with the T-cell receptor), (Margalit et al., Computer Prediction of T-cell Epitopes, New Generation Vaccines Marcel-Dekker, Inc, ed. G. C. Woodrow et al., (1990) pp. 109-116) and further that the amphipathic structures have an α-helical configuration (see, e.g., Spouge et al., J. Immunol. (1987) 138:204-212; Berkower et al., J. Immunol. (1986) 136:2498-2503).
Hence, segments of proteins that include T-cell epitopes can be readily predicted using numerous computer programs. (See e.g., Margalit et al., Computer Prediction of T-cell Epitopes, New Generation Vaccines Marcel-Dekker, Inc, ed. G. C. Woodrow et al., (1990) pp. 109-116). Such programs generally compare the amino acid sequence of a peptide to sequences known to induce a T-cell response, and search for patterns of amino acids which are believed to be required for a T-cell epitope.
An “immunological response” to an HCV antigen (including both polypeptide and polynucleotides encoding polypeptides that are expressed in vivo) or composition is the development in a subject of a humoral and/or a cellular immune response to molecules present in the composition of interest. For purposes of the present invention, a “humoral immune response” refers to an immune response mediated by antibody molecules, while a “cellular immune response” is one mediated by T-lymphocytes and/or other white blood cells. One important aspect of cellular immunity involves an antigen-specific response by cytolytic T-cells (“CTLs”). CTLs have specificity for peptide antigens that are presented in association with proteins encoded by the major histocompatibility complex (MHC) and expressed on the surfaces of cells. CTLs help induce and promote the intracellular destruction of intracellular microbes, or the lysis of cells infected with such microbes. Another aspect of cellular immunity involves an antigen-specific response by helper T-cells. Helper T-cells act to help stimulate the function, and focus the activity of, nonspecific effector cells against cells displaying peptide antigens in association with MHC molecules on their surface. A “cellular immune response” also refers to the production of cytokines, chemokines and other such molecules produced by activated T-cells and/or other white blood cells, including those derived from CD4+ and CD8+ T-cells.
A composition or vaccine that elicits a cellular immune response may serve to sensitize a vertebrate subject by the presentation of antigen in association with MHC molecules at the cell surface. The cell-mediated immune response is directed at, or near, cells presenting antigen at their surface. In addition, antigen-specific T-lymphocytes can be generated to allow for the future protection of an immunized host.
The ability of a particular antigen to stimulate a cell-mediated immunological response may be determined by a number of assays, such as by lymphoproliferation (lymphocyte activation) assays, CTL cytotoxic cell assays, or by assaying for T-lymphocytes specific for the antigen in a sensitized subject. Such assays are well known in the art. See, e.g., Erickson et al., J. Immunol. (1993) 151:4189-4199; Doe et al., Eur. J. Immunol. (1994) 24:2369-2376; and the examples below.
Thus, an immunological response as used herein may be one which stimulates the production of CTLs, and/or the production or activation of helper T-cells. The antigen of interest may also elicit an antibody-mediated immune response. Hence, an immunological response may include one or more of the following effects: the production of antibodies by B-cells; and/or the activation of suppressor T-cells and/or γδ T-cells directed specifically to an antigen or antigens present in the composition or vaccine of interest. These responses may serve to neutralize infectivity, and/or mediate antibody-complement, or antibody dependent cell cytotoxicity (ADCC) to provide protection or alleviation of symptoms to an immunized host. Such responses can be determined using standard immunoassays and neutralization assays, well known in the art.
By “equivalent antigenic determinant” is meant an antigenic determinant from different sub-species or strains of HCV, such as from strains 1, 2, 3, etc., of HCV which antigenic determinants are not necessarily identical due to sequence variation, but which occur in equivalent positions in the HCV sequence in question. In general the amino acid sequences of equivalent antigenic determinants will have a high degree of sequence homology, e.g., amino acid sequence homology of more than 30%, usually more than 40%, such as more than 60%, and even more than 80-90% homology, when the two sequences are aligned.
A “coding sequence” or a sequence which “encodes” a selected polypeptide, is a nucleic acid molecule which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5′ (amino) terminus and a translation stop codon at the 3′ (carboxy) terminus. A transcription termination sequence may be located 3′ to the coding sequence.
A “nucleic acid” molecule or “polynucleotide” can include both double- and single-stranded sequences and refers to, but is not limited to, cDNA from viral, procaryotic or eucaryotic mRNA, genomic DNA sequences from viral (e.g. DNA viruses and retroviruses) or procaryotic DNA, and especially synthetic DNA sequences. The term also captures sequences that include any of the known base analogs of DNA and RNA.
An “HCV polynucleotide” is a polynucleotide that encodes an HCV polypeptide, as defined above.
“Operably linked” refers to an arrangement of elements wherein the components so described are configured so as to perform their desired function. Thus, a given promoter operably linked to a coding sequence is capable of effecting the expression of the coding sequence when the proper transcription factors, etc., are present. The promoter need not be contiguous with the coding sequence, so long as it functions to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between the promoter sequence and the coding sequence, as can transcribed introns, and the promoter sequence can still be considered “operably linked” to the coding sequence.
“Recombinant” as used herein to describe a nucleic acid molecule means a polynucleotide of genomic, cDNA, viral, semisynthetic, or synthetic origin which, by virtue of its origin or manipulation is not associated with all or a portion of the polynucleotide with which it is associated in nature. The term “recombinant” as used with respect to a protein or polypeptide means a polypeptide produced by expression of a recombinant polynucleotide. In general, the gene of interest is cloned and then expressed in transformed organisms, as described further below. The host organism expresses the foreign gene to produce the protein under expression conditions.
A “control element” refers to a polynucleotide sequence which aids in the expression of a coding sequence to which it is linked. The term includes promoters, transcription termination sequences, upstream regulatory domains, polyadenylation signals, untranslated regions, including 5′-UTRs and 3′-UTRs and when appropriate, leader sequences and enhancers, which collectively provide for the transcription and translation of a coding sequence in a host cell.
A “promoter” as used herein is a DNA regulatory region capable of binding RNA polymerase in a host cell and initiating transcription of a downstream (3′ direction) coding sequence operably linked thereto. For purposes of the present invention, a promoter sequence includes the minimum number of bases or elements necessary to initiate transcription of a gene of interest at levels detectable above background. Within the promoter sequence is a transcription initiation site, as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. Eucaryotic promoters will often, but not always, contain “TATA” boxes and “CAT” boxes.
A control sequence “directs the transcription” of a coding sequence in a cell when RNA polymerase will bind the promoter sequence and transcribe the coding sequence into mRNA, which is then translated into the polypeptide encoded by the coding sequence.
“Expression cassette” or “expression construct” refers to an assembly which is capable of directing the expression of the sequence(s) or gene(s) of interest. The expression cassette includes control elements, as described above, such as a promoter which is operably linked to (so as to direct transcription of) the sequence(s) or gene(s) of interest, and often includes a polyadenylation sequence as well. Within certain embodiments of the invention, the expression cassette described herein may be contained within a plasmid construct. In addition to the components of the expression cassette, the plasmid construct may also include, one or more selectable markers, a signal which allows the plasmid construct to exist as single-stranded DNA (e.g., a M13 origin of replication), at least one multiple cloning site, and a “mammalian” origin of replication (e.g., a SV40 or adenovirus origin of replication).
“Transformation,” as used herein, refers to the insertion of an exogenous polynucleotide into a host cell, irrespective of the method used for insertion: for example, transformation by direct uptake, transfection, infection, and the like. For particular methods of transfection, see further below. The exogenous polynucleotide may be maintained as a nonintegrated vector, for example, an episome, or alternatively, may be integrated into the host genome.
A “host cell” is a cell which has been transformed, or is capable of transformation, by an exogenous DNA sequence.
By “isolated” is meant, when referring to a polypeptide, that the indicated molecule is separate and discrete from the whole organism with which the molecule is found in nature or is present in the substantial absence of other biological macro-molecules of the same type. The term “isolated” with respect to a polynucleotide is a nucleic acid molecule devoid, in whole or part, of sequences normally associated with it in nature; or a sequence, as it exists in nature, but having heterologous sequences in association therewith; or a molecule disassociated from the chromosome.
The term “purified” as used herein preferably means at least 75% by weight, more preferably at least 85% by weight, more preferably still at least 95% by weight, and most preferably at least 98% by weight, of biological macromolecules of the same type are present.
“Homology” refers to the percent identity between two polynucleotide or two polypeptide moieties. Two DNA, or two polypeptide sequences are “substantially homologous” to each other when the sequences exhibit at least about 50%, preferably at least about 75%, more preferably at least about 80%-85%, preferably at least about 90%, and most preferably at least about 95%-98%, or more, sequence identity over a defined length of the molecules. As used herein, substantially homologous also refers to sequences showing complete identity to the specified DNA or polypeptide sequence.
In general, “identity” refers to an exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Percent identity can be determined by a direct comparison of the sequence information between two molecules by aligning the sequences, counting the exact number of matches between the two aligned sequences, dividing by the length of the shorter sequence, and multiplying the result by 100. Readily available computer programs can be used to aid in the analysis, such as ALIGN, Dayhoff, M. O. in Atlas of Protein Sequence and Structure M. O. Dayhoff ed., 5 Suppl. 3:353-358, National biomedical Research Foundation, Washington, D.C., which adapts the local homology algorithm of Smith and Waterman Advances in Appl. Math. 2:482-489, 1981 for peptide analysis. Programs for determining nucleotide sequence identity are available in the Wisconsin Sequence Analysis Package, Version 8 (available from Genetics Computer Group, Madison, Wis.) for example, the BESTFIT, FASTA and GAP programs, which also rely on the Smith and Waterman algorithm. These programs are readily utilized with the default parameters recommended by the manufacturer and described in the Wisconsin Sequence Analysis Package referred to above. For example, percent identity of a particular nucleotide sequence to a reference sequence can be determined using the homology algorithm of Smith and Waterman with a default scoring table and a gap penalty of six nucleotide positions.
Another method of establishing percent identity in the context of the present invention is to use the MPSRCH package of programs copyrighted by the University of Edinburgh, developed by John F. Collins and Shane S. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View, Calif.). From this suite of packages the Smith-Waterman algorithm can be employed where default parameters are used for the scoring table (for example, gap open penalty of 12, gap extension penalty of one, and a gap of six). From the data generated the “Match” value reflects “sequence identity.” Other suitable programs for calculating the percent identity or similarity between sequences are generally known in the art, for example, another alignment program is BLAST, used with default parameters. For example, BLASTN and BLASTP can be used using the following default parameters: genetic code=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swiss protein+Spupdate+PIR. Details of these programs can be found at the following internet address: http://www.ncbi.nln.gov/cgi-bin/BLAST.
Alternatively, homology can be determined by hybridization of polynucleotides under conditions which form stable duplexes between homologous regions, followed by digestion with single-stranded-specific nuclease(s), and size determination of the digested fragments. DNA sequences that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Sambrook et al., supra; DNA Cloning, supra; Nucleic Acid Hybridization, supra.
By “nucleic acid immunization” is meant the introduction of a nucleic acid molecule encoding one or more selected antigens into a host cell, for the in vivo expression of the antigen or antigens. The nucleic acid molecule can be introduced directly into the recipient subject, such as by injection, inhalation, oral, intranasal and mucosal administration, or the like, or can be introduced ex vivo, into cells which have been removed from the host. In the latter case, the transformed cells are reintroduced into the subject where an immune response can be mounted against the antigen encoded by the nucleic acid molecule.
As used herein, “treatment” refers to any of (i) the prevention of infection or reinfection, as in a traditional vaccine, (ii) the reduction or elimination of symptoms, and (iii) the substantial or complete elimination of the pathogen in question. Treatment may be effected prophylactically (prior to infection) or therapeutically (following infection).
By “vertebrate subject” is meant any member of the subphylum cordata, including, without limitation, humans and other primates, including non-human primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs; birds, including domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like. The term does not denote a particular age. Thus, both adult and newborn individuals are intended to be covered. The invention described herein is intended for use in any of the above vertebrate species, since the immune systems of all of these vertebrates operate similarly.
II. MODES OF CARRYING OUT THE INVENTIONBefore describing the present invention in detail, it is to be understood that this invention is not limited to particular formulations or process parameters as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting.
Although a number of compositions and methods similar or equivalent to those described herein can be used in the practice of the present invention, the preferred materials and methods are described herein.
It is a discovery of the present invention that fusion proteins, combinations of the individual components of these fusions, and polynucleotides encoding the same, comprising an NS3, an NS4, and an NS5a polypeptide with or without a core polypeptide, or an NS3, an NS4, an NS5a, and an NS5b polypeptide, with or without a core polypeptide, of an HCV virus can be used to activate HCV-specific T cells, i.e., T cells which recognize epitopes of these polypeptides.
The present invention also pertains to compositions comprising HCV nonstructural fusion proteins and HCV E1E2 complexes, as well as compositions comprising polynucleotides encoding the same or combinations of polypeptides and polynucleotides.
The proteins, polynucleotides, compositions and combinations of the present invention can be used to stimulate a cellular immune response, such as to activate HCV-specific T cells, i.e., T cells which recognize epitopes of these polypeptides. Activation of HCV-specific T cells provides both in vitro and in vivo model systems for the development of HCV vaccines, particularly for identifying HCV polypeptide epitopes associated with a response. The compositions can also be used to generate an immune response against HCV in a mammal, particularly a CTL response for either therapeutic or prophylactic purposes.
Fusion ProteinsThe genomes of HCV strains contain a single open reading frame of approximately 9,000 to 12,000 nucleotides, which is transcribed into a polyprotein. As shown in
Fusion proteins for use in the compositions and methods, and polynucleotides encoding therefor, include or encode an NS3 polypeptide, an NS4 (NS4a and/or NS4b) polypeptide, an NS5a polypeptide and, optionally, an NS5b polypeptide. The fusion proteins may or may not include all or part of the core region. In certain embodiments, none of the core region is present in the compositions. The nonstructural regions need not be in the order in which they naturally occur in the native HCV polyprotein. Thus, for example, the NS5b polypeptide may be at the N- and/or C-terminus of the fusion or may be found internally. These polypeptides may be derived from the same HCV isolate, or from different strains and isolates including isolates having any of the various HCV genotypes, to provide increased protection against a broad range of HCV genotypes. Additionally, polypeptides can be selected based on the particular viral clades endemic in specific geographic regions where vaccine compositions containing the fusions will be used. It is readily apparent that the subject fusions provide an effective means of treating HCV infection in a wide variety of contexts.
In one embodiment, the fusion protein of the present invention includes an NS3 polypeptide that has been modified to inhibit protease activity, such that further cleavage of the fusion is inhibited. The NS3 polypeptide can be modified by deletion of all or a portion of the NS3 protease domain. Alternatively, proteolytic activity can be inhibited by substitutions of amino acids within active regions of the protease domain. Finally, additions of amino acids to active regions of the domain, such that the catalytic site is modified, will also serve to inhibit proteolytic activity.
As explained above, the protease activity is found at about amino acid positions 1027-1207, numbered relative to the full-length HCV-1 polyprotein (see, Choo et al., Proc. Natl. Acad. Sci. USA (1991) 88:2451-2455), positions 2-182 of
For example, protease activity or lack thereof may be determined using assays well known in the art. See, e.g., Takeshita et al., Anal. Biochem. (1997) 247:242-246; Kakiuchi et al., J. Biochem. (1997) 122:749-755; SalI et al., Biochemistry (1998) 37:3392-3401; Cho et al., J. Virol. Meth. (1998) 72:109-115; Cerretani et al., Anal. Biochem. (1999) 266:192-197; Zhang et al., Anal. Biochem. (1999) 270:268-275; Kakiuchi et al., J. Virol. Meth. (1999) 80:77-84; Fowler et al., J. Biomol. Screen. (2000) 5:153-158; and Kim et al., Anal. Biochem. (2000) 284:42-48.
The NS3; NS4; NS5a, and NS5b polypeptides present in the various fusions described above can either be full-length polypeptides or portions of NS3, NS4 (NS4a and/or NS4b), NS5a, and NS5b polypeptides. The portions of NS3, NS4, NS5a, and NS5b polypeptides making up the fusion protein preferably comprise at least one epitope, which is recognized by a T cell receptor on an activated T cell, such as 2152-HEYPVGSQL-2160 (SEQ ID NO: 1) and/or 2224-AELIEANLLWRQEMG-2238 (SEQ ID NO:2). Epitopes of NS3, NS4 (NS4a and NS4b), NS5a, NS5b, NS3NS4NS5a, and NS3NS4NS5aNS5b can be identified by several methods. For example, NS3, NS4, NS5a, NS5b polypeptides or fusion proteins comprising any combination of the above, can be isolated, for example, by immunoaffinity purification using a monoclonal antibody for the polypeptide or protein. The isolated protein sequence can then be screened by preparing a series of short peptides by proteolytic cleavage of the purified protein, which together span the entire protein sequence. By starting with, for example, 100-mer polypeptides, each polypeptide can be tested for the presence of epitopes recognized by a T-cell receptor on an HCV-activated T cell, progressively smaller and overlapping fragments can then be tested from an identified 100-mer to map the epitope of interest.
Epitopes recognized by a T-cell receptor on an HCV-activated T cell can be identified by, for example, 51Cr release assay or by lymphoproliferation assay (see the examples). In a 51Cr release assay, target cells can be constructed that display the epitope of interest by cloning a polynucleotide encoding the epitope into an expression vector and transforming the expression vector into the target cells. HCV-specific CD8+ T cells will lyse target cells displaying, for example, an NS3, NS4, NS5a, NS5b, NS3NS4NS5a, or NS3NS4NS5aNS5b epitope and will not lyse cells that do not display such an epitope. In a lymphoproliferation assay, HCV-activated CD4+ T cells will proliferate when cultured with, for example, an NS3, NS4, NS5a, NS5b, NS3NS4NS5a, or NS3NS4NS5aNS5b epitopic peptide, but not in the absence of an HCV epitopic peptide.
NS3, NS4, NS5a, and NS5b polypeptides can occur in any order in the fusion protein. If desired, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more of one or more of the polypeptides may occur in the fusion protein. Multiple viral strains of HCV occur, and NS3, NS4, NS5a, and NS5b polypeptides of any of these strains can be used in a fusion protein. A representative fusion protein for use in the present invention is shown if
Nucleic acid and amino acid sequences of a number of HCV strains and isolates, including nucleic acid and amino acid sequences of NS3, NS4, NS5a, NS5b genes and polypeptides have been determined. For example, isolate HCV J1.1 is described in Kubo et al. (1989) Japan. Nucl. Acids Res. 17:10367-10372; Takeuchi et al. (1990) Gene 91:287-291; Takeuchi et al. (1990) J. Gen. Virol. 71:3027-3033; and Takeuchi et al. (1990) Nucl. Acids Res. 18:4626. The complete coding sequences of two independent isolates, HCV-J and BK, are described by Kato et al., (1990) Proc. Natl. Acad. Sci. USA 87:9524-9528 and Takamizawa et al., (1991) J. Virol. 65:1105-1113 respectively.
Publications that describe HCV-1 isolates include Choo et al. (1990) Brit. Med. Bull. 46:423-441; Choo et al. (1991) Proc. Natl. Acad. Sci. USA 88:2451-2455 and Han et al. (1991) Proc. Natl. Acad. Sci. USA 88:1711-1715. HCV isolates HC-J1 and HC-J4 are described in Okamoto et al. (1991) Japan J. Exp. Med. 60:167-177. HCV isolates HCT 18˜, HCT 23, Th, HCT 27, EC1 and EC10 are described in Weiner et al. (1991) Virol. 180:842-848. HCV isolates Pt-1, HCV-K1 and HCV-K2 are described in Enomoto et al. (1990) Biochem. Biophys. Res. Commun. 170:1021-1025. HCV isolates A, C, D & E are described in Tsukiyama-Kohara et al. (1991) Virus Genes 5:243-254.
Each of the NS3, NS4, NS5a, and NS5b components of a fusion protein can be obtained from the same HCV strain or isolate or from different HCV strains or isolates. Fusion proteins comprising HCV polypeptides from, for example, the NS3 polypeptide can be derived from a first strain of HCV, and the NS4, and NS5a polypeptides can be derived from a second strain of HCV. Alternatively, the NS4 polypeptide can be derived from a first strain of HCV, and the NS3 and NS5a polypeptides can be derived from a second strain of HCV. Optionally, the NS5a polypeptide can be derived from a first strain of HCV, and the NS3 and NS4 polypeptides can be derived from a second strain of HCV. NS3, NS4 and NS5a polypeptides that are each derived from different HCV strains can also be used in an HCV fusion protein. Similarly, in a fusion protein comprising NS5b, at least one of the NS3, NS4, NS5a, and NS5b polypeptides can be derived from a different HCV strain than the other polypeptides. Optionally, NS3, NS4, NS5a, and NS5b polypeptides that are each derived from different HCV strains can also be used in an NS3NS4NS5aNS5b fusion protein.
In addition to NS3, NS4a, NS4b, NS5a and NS5b, the fusion proteins can contain other polypeptides derived from the HCV polyprotein. For example, it may be desirable to include polypeptides derived from the core region of the HCV polyprotein. This region occurs at amino acid positions 1-191 of the HCV polyprotein, numbered relative to HCV-1. Either the full-length protein, fragments thereof, such as amino acids 1-150, e.g., amino acids 1-130, 1-120, for example, amino acids 1-121, 1-122, 1-123, etc., or smaller fragments containing epitopes of the full-length protein may be used in the subject fusions, such as those epitopes found between amino acids 10-53, amino acids 10-45, amino acids 67-88, amino acids 120-130, or any of the core epitopes identified in, e.g., Houghton et al., U.S. Pat. No. 5,350,671; Chien et al., Proc. Natl. Acad. Sci. USA (1992) 89:10011-10015; Chien et al., J. Gastroent. Hepatol. (1993) 8:S33-39; Chien et al., International Publication No. WO 93/00365; Chien, D. Y., International Publication No. WO 94/01778; and U.S. Pat. Nos. 6,280,927 and 6,150,087, the disclosures of which are incorporated herein by reference in their entireties. Moreover, a protein resulting from a frameshift in the core region of the polyprotein, such as described in International Publication No. WO 99/63941, may be used. The fusions may also contain polynucleotides encoding E1E2 polypeptides, as described further below.
Preferably, the above-described fusion proteins, as well as the individual components of these proteins, are produced recombinantly. A polynucleotide encoding these proteins can be introduced into an expression vector which can be expressed in a suitable expression system. A variety of bacterial, yeast, mammalian and insect expression systems are available in the art and any such expression system can be used. Optionally, a polynucleotide encoding these proteins can be translated in a cell-free translation system. Such methods are well known in the art. The proteins also can be constructed by solid phase protein synthesis.
If desired, the fusion proteins, or the individual components of these proteins, also can contain other non-HCV amino acid sequences, such as amino acid linkers or signal sequences, as well as ligands useful in protein purification, such as glutathione-S-transferase and staphylococcal protein A.
E1E2 PolypeptidesAs explained above, the compositions of the present invention may also include E1 and E2 polypeptides, complexes of these polypeptides or polynucleotides encoding the same. The E1 and E2 polypeptides and complexes thereof can be provided independent of the nonstructural fusion protein or can be incorporated into the same fusion. Moreover, E1E2 complexes can be provided as proteins, or as polynucleotides encoding the same.
In this regard, E1, E2 and p7 are known to contain human T-cell epitopes (both CD4+ and CD8+) and including one or more of these epitopes serves to increase vaccine efficacy as well as to increase protective levels against multiple HCV genotypes. Moreover, multiple copies of specific, conserved T-cell epitopes can also be used in E1E2 complexes, such as a composite of epitopes from different genotypes.
As explained above, the E1 and E2 polypeptides that make up the E1E2 complexes can be associated either through non-covalent or covalent interactions. Such complexes may be made up of immunogenic fragments of E1 and E2 which comprise epitopes. For example, fragments of E1 polypeptides can comprise from about 5 to nearly the full-length of the molecule, such as 6, 10, 25, 50, 75, 100, 125, 150, 175, 185 or more amino acids of an E1 polypeptide, or any integer between the stated numbers. Similarly, fragments of E2 polypeptides can comprise 6, 10, 25, 50, 75, 100, 150, 200, 250, 300, or 350 amino acids of an E2 polypeptide, or any integer between the stated numbers. The E1 and E2 polypeptides may be from the same or different HCV strains. For example, epitopes derived from, e.g., the hypervariable region of E2, such as a region spanning amino acids 384-410 or 390-410, can be included in the E2 polypeptide. A particularly effective E2 epitope to incorporate into the E2 sequence or E1E2 complexes is one which includes a consensus sequence derived from this region, such as the consensus sequence Gly-Ser-Ala-Ala-Arg-Thr-Thr-Ser-Gly-Phe-Val-Ser-Leu-Phe-Ala-Pro-Gly-Ala-Lys-Gln-Asn (SEQ ID NO:5), which represents a consensus sequence for amino acids 390-410 of the HCV type 1 genome. Additional epitopes of E1 and E2 are known and described in, e.g., Chien et al., International Publication No. WO 93/00365, incorporated by reference herein in its entirety.
Moreover, the E1 and E2 polypeptides may lack all or a portion of the membrane spanning domain. The membrane anchor sequence functions to associate the polypeptide to the endoplasmic reticulum. Normally, such polypeptides are capable of secretion into growth medium in which an organism expressing the protein is cultured. However, as described in International Publication No. WO 98/50556, such polypeptides may also be recovered intracellularly. Secretion into growth medium is readily determined using a number of detection techniques, including, e.g., polyacrylamide gel electrophoresis and the like, and immunological techniques such as immunoprecipitation assays as described in, e.g., International Publication No. WO 96/04301, published Feb. 15, 1996. With E1, generally polypeptides terminating with about amino acid position 370 and higher (based on the numbering of HCV1 E1) will be retained by the ER and hence not secreted into growth media. With E2, polypeptides terminating with about amino acid position 731 and higher (also based on the numbering of the HCV1 E2 sequence) will be retained by the ER and not secreted. (See, e.g., International Publication No. WO 96/04301, published Feb. 15, 1996). It should be noted that these amino acid positions are not absolute and may vary to some degree. Thus, the present invention contemplates the use of E1 and E2 polypeptides which retain the transmembrane binding domain, as well as polypeptides which lack all or a portion of the transmembrane binding domain, including E1 polypeptides terminating at about amino acids 369 and lower, and E2 polypeptides, terminating at about amino acids 730 and lower, are intended to be captured by the present invention. Furthermore, the C-terminal truncation can extend beyond the transmembrane spanning domain towards the N-terminus. Thus, for example, E1 truncations occurring at positions lower than, e.g., 360 and E2 truncations occurring at positions lower than, e.g., 715, are also encompassed by the present invention. All that is necessary is that the truncated E1 and E2 polypeptides remain functional for their intended purpose. However, particularly preferred truncated E1 constructs are those that do not extend beyond about amino acid 300. Most preferred are those terminating at position 360. Preferred truncated E2 constructs are those with C-terminal truncations that do not extend beyond about amino acid position 715. Particularly preferred E2 truncations are those molecules truncated after any of amino acids 715-730, such as 725. If truncated molecules are used, it is preferable to use E1 and E2 molecules that are both truncated.
E2 exists as multiple species (Spaete et al., Virol. (1992) 188:819-830; Selby et al., J. Virol. (1996) 70:5177-5182; Grakoui et al., J. Virol. (1993) 67:1385-1395; Tomei et al., J. Virol. (1993) 67:4017-4026) and clipping and proteolysis may occur at the N- and C-termini of the E1 and E2 polypeptides. Thus, an E2 polypeptide for use herein may comprise at least amino acids 405-661, e.g., 400,401, 402 . . . to 661, such as 384-661, 384-715, 384-746, 384-749 or 384-809, or 384 to any C-terminus between 661-809, of an HCV polyprotein, numbered relative to the full-length HCV-1 polyprotein. Similarly, preferable E1 polypeptides for use herein can comprise amino acids 192-326, 192-330, 192-333, 192-360, 192-363, 192-383, or 192 to any C-terminus between 326-383, of an HCV polyprotein.
The E1 and E2 polypeptides and complexes thereof may also be present as asialoglycoproteins. Such asialoglycoproteins are produced by methods known in the art, such as by using cells in which terminal glycosylation is blocked. When these proteins are expressed in such cells and isolated by GNA lectin affinity chromatography, the E1 and E2 proteins aggregate spontaneously. Detailed methods for producing these E1E2 aggregates are described in, e.g., U.S. Pat. No. 6,074,852, incorporated herein by reference in its entirety. For example, E1E2 complexes are readily produced recombinantly, either as fusion proteins or by e.g., co-transfecting host cells with constructs encoding for the E1 and E2 polypeptides of interest. Co-transfection can be accomplished either in trans or cis, i.e., by using separate vectors or by using a single vector which bears both of the E1 and E2 genes. If done using a single vector, both genes can be driven by a single set of control elements or, alternatively, the genes can be present on the vector in individual expression cassettes, driven by individual control elements. Following expression, the E1 and E2 proteins will spontaneously associate. Alternatively, the complexes can be formed by mixing the individual proteins together which have been produced separately, either in purified or semi-purified form, or even by mixing culture media in which host cells expressing the proteins, have been cultured, if the proteins are secreted. Finally, the E1E2 complexes of the present invention may be expressed as a fusion protein wherein the desired portion of E1 is fused to the desired portion of E2.
Moreover, the E1E2 complexes may be present as a heterogeneous mixture of molecules, due to clipping and proteolytic cleavage, as described above. Thus, a composition including E1E2 complexes may include multiple species of E1E2, such as E1E2 terminating at amino acid 746 (E1E2746), E1E2 terminating at amino acid 809 (E1E2809), or any of the other various E1 and E2 molecules described above, such as E2 molecules with N-terminal truncations of from 1-20 amino acids, such as E2 species beginning at amino acid 387, amino acid 402, amino acid 403, etc.
E1E2 complexes are readily produced recombinantly, either as fusion proteins or by e.g., co-transfecting host cells with constructs encoding for the E1 and E2 polypeptides of interest. Co-transfection can be accomplished either in trans or cis, i.e., by using separate vectors or by using a single vector which bears both of the E1 and E2 genes. If done using a single vector, both genes can be driven by a single set of control elements or, alternatively, the genes can be present on the vector in individual expression cassettes, driven by individual control elements. Following expression, the E1 and E2 proteins will spontaneously associate. Alternatively, the complexes can be formed by mixing the individual proteins together which have been produced separately, either in purified or semi-purified form, or even by mixing culture media in which host cells expressing the proteins, have been cultured, if the proteins are secreted. Finally, the E1E2 complexes of the present invention may be expressed as a fusion protein wherein the desired portion of E1 is fused to the desired portion of E2.
Methods for producing E1E2 complexes from full-length, truncated E1 and E2 proteins which are secreted into media, as well as intracellularly produced truncated proteins, are known in the art. For example, such complexes may be produced recombinantly, as described in U.S. Pat. No. 6,121,020; Ralston et al., J. Virol. (1993) 67:6753-6761, Grakoui et al., J Virol. (1993) 67:1385-1395; and Lanford et al., Virology (1993) 197:225-235.
Polynucleotides Encoding the Fusion Proteins and E1E2 ComplexesPolynucleotides contain less than an entire HCV genome and can be RNA or single- or double-stranded DNA. Preferably, the polynucleotides are isolated free of other components, such as proteins and lipids. The polynucleotides encode the fusion proteins, E1 and E2 polypeptides and complexes thereof, described above, and thus comprise coding sequences thereof. Polynucleotides of the invention can also comprise other non-HCV nucleotide sequences, such as sequences coding for linkers, signal sequences, or ligands useful in protein purification such as glutathione-S-transferase and staphylococcal protein A.
Polynucleotides encoding the various HCV polypeptides can be isolated from a genomic library derived from nucleic acid sequences present in, for example, the plasma, serum, or liver homogenate of an HCV infected individual or can be synthesized in the laboratory, for example, using an automatic synthesizer. An amplification method such as PCR can be used to amplify polynucleotides from either HCV genomic DNA or cDNA encoding therefor.
Polynucleotides can comprise coding sequences for these polypeptides which occur naturally or can include artificial sequences which do not occur in nature. These polynucleotides can be ligated to form a coding sequence for the fusion proteins and E1E2 complexes using standard molecular biology techniques. If desired, polynucleotides can be cloned into an expression vector and transformed into, for example, bacterial, yeast, insect, or mammalian cells so that the fusion proteins of the invention can be expressed in and isolated from a cell culture.
The expression constructs of the present invention, including the desired fusion, or individual expression constructs comprising the individual components of these fusions, may be used for nucleic acid immunization, to stimulate an immunological response, such as a cellular immune response, using standard gene delivery protocols. Methods for gene delivery are known in the art. See, e.g., U.S. Pat. Nos. 5,399,346, 5,580,859, 5,589,466, incorporated by reference herein in their entireties. Genes can be delivered either directly to the vertebrate subject or, alternatively, delivered ex vivo, to cells derived from the subject and the cells reimplanted in the subject. For example, the constructs can be delivered as plasmid DNA, e.g., contained within a plasmid, such as pBR322, pUC, or ColE1
Additionally, the expression constructs can be packaged in liposomes prior to delivery to the cells. Lipid encapsulation is generally accomplished using liposomes which are able to stably bind or entrap and retain nucleic acid. The ratio of condensed DNA to lipid preparation can vary but will generally be around 1:1 (mg DNA:micromoles lipid), or more of lipid. For a review of the use of liposomes as carriers for delivery of nucleic acids, see, Hug and Sleight, Biochim. Biophys. Acta. (1991) 1097:1-17; Straubinger et al., in Methods of Enzymology (1983), Vol. 101, pp. 512-527.
Liposomal preparations for use with the present invention include cationic (positively charged), anionic (negatively charged) and neutral preparations, with cationic liposomes particularly preferred. Cationic liposomes are readily available. For example, N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes are available under the trademark Lipofectin, from GIBCO BRL, Grand Island, N.Y. (See, also, Felgner et al., Proc. Natl. Acad. Sci. USA (1987) 84:7413-7416). Other commercially available lipids include transfectace (DDAB/DOPE) and DOTAP/DOPE (Boerhinger). Other cationic liposomes can be prepared from readily available materials using techniques well known in the art. See, e.g., Szoka et al., Proc. Natl. Acad. Sci. USA (1978) 75:4194-4198; PCT Publication No. WO 90/11092 for a description of the synthesis of DOTAP (1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes. The various liposome-nucleic acid complexes are prepared using methods known in the art. See, e.g., Straubinger et al., in METHODS OF IMMUNOLOGY (1983), Vol. 101, pp. 512-527; Szoka et al., Proc. Natl. Acad. Sci. USA (1978) 75:4194-4198; Papahadjopoulos et al., Biochim. Biophys. Acta (1975) 394:483; Wilson et al., Cell (1979) 17:77); Deamer and Bangham, Biochim. Biophys. Acta (1976) 443:629; Ostro et al., Biochem. Biophys. Res. Commun. (1977) 76:836; Fraley et al., Proc. Natl. Acad. Sci. USA (1979) 76:3348); Enoch and Strittmatter, Proc. Natl. Acad. Sci. USA (1979) 76:145); Fraley et al., J. Biol. Chem. (1980) 255:10431; Szoka and Papahadjopoulos, Proc. Natl. Acad. Sci. USA (1978) 75:145; and Schaefer-Ridder et al., Science (1982) 215:166.
The DNA can also be delivered in cochleate lipid compositions similar to those described by Papahadjopoulos et al., Biochem. Biophys. Acta. (1975) 394:483-491. See, also, U.S. Pat. Nos. 4,663,161 and 4,871,488.
A number of viral based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems, such as murine sarcoma virus, mouse mammary tumor virus, Moloney murine leukemia virus, and Rous sarcoma virus. A selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo. A number of retroviral systems have been described (U.S. Pat. No. 5,219,740; Miller and Rosman, BioTechniques (1989) 7:980-990; Miller, A. D., Human Gene Therapy (1990) 1:5-14; Scarpa et al., Virology (1991) 180:849-852; Burns et al., Proc. Natl. Acad. Sci. USA (1993) 90:8033-8037; and Boris-Lawrie and Temin, Cur. Opin. Genet. Develop. (1993) 3:102-109. Briefly, retroviral gene delivery vehicles of the present invention may be readily constructed from a wide variety of retroviruses, including for example, B, C, and D type retroviruses as well as spumaviruses and lentiviruses such as FIV, HIV, HIV-1, HIV-2 and SIV (see RNA Tumor Viruses, Second Edition, Cold Spring Harbor Laboratory, 1985). Such retroviruses may be readily obtained from depositories or collections such as the American Type Culture Collection (“ATCC”; 10801 University Blvd., Manassas, Va. 20110-2209), or isolated from known sources using commonly available techniques.
A number of adenovirus vectors have also been described, such as adenovirus Type 2 and Type 5 vectors. Unlike retroviruses which integrate into the host genome, adenoviruses persist extrachromosomally thus minimizing the risks associated with insertional mutagenesis (Haj-Ahmad and Graham, J. Virol. (1986) 57:267-274; Bett et al., J. Virol. (1993) 67:5911-5921; Mittereder et al., Human Gene Therapy (1994) 5:717-729; Seth et al., J. Virol. (1994) 68:933-940; Barr et al., Gene Therapy (1994) 1:51-58; Berkner, K. L. BioTechniques (1988) 6:616-629; and Rich et al., Human Gene Therapy (1993) 4:461-476).
Molecular conjugate vectors, such as the adenovirus chimeric vectors described in Michael et al., J. Biol. Chem. (1993) 268:6866-6869 and Wagner et al., Proc. Natl. Acad. Sci. USA (1992) 89:6099-6103, can also be used for gene delivery.
Members of the Alphavirus genus, such as but not limited to vectors derived from the Sindbis and Semliki Forest viruses, VEE, will also find use as viral vectors for delivering the gene of interest. For a description of Sindbis-virus derived vectors useful for the practice of the instant methods, see, Dubensky et al., J. Virol. (1996) 70:508-519; and International Publication Nos. WO 95/07995 and WO 96/17072.
Other vectors can be used, including but not limited to simian virus 40 and cytomegalovirus. Bacterial vectors, such as Salmonella ssp. Yersinia enterocolitica, Shigella spp., Vibrio cholerae, Mycobacterium strain BCG, and Listeria monocytogenes can be used. Minichromosomes such as MC and MC1, bacteriophages, cosmids (plasmids into which phage lambda cos sites have been inserted) and replicons (genetic elements that are capable of replication under their own control in a cell) can also be used.
The expression constructs may also be encapsulated, adsorbed to, or associated with, particulate carriers. Such carriers present multiple copies of a selected molecule to the immune system and promote trapping and retention of molecules in local lymph nodes. The particles can be phagocytosed by macrophages and can enhance antigen presentation through cytokine release. Examples of particulate carriers include those derived from polymethyl methacrylate polymers, as well as microparticles derived from poly(lactides) and poly(lactide-co-glycolides), known as PLG. See, e.g., Jeffery et al., Pharm. Res. (1993) 10:362-368; and McGee et al., J. Microencap. (1996).
One preferred method for adsorbing macromolecules onto prepared microparticles is described in International Publication No. WO 00/050006, incorporated herein by reference in its entirety. Briefly, microparticles are rehydrated and dispersed to an essentially monomeric suspension of microparticles using dialyzable anionic or cationic detergents. Useful detergents include, but are not limited to, any of the various N-methylglucamides (known as MEGAs), such as heptanoyl-N-methylglucamide (MEGA-7), octanoyl-N-methylglucamide (MEGA-8), nonanoyl-N-methylglucamide (MEGA-9), and decanoyl-N-methyl-glucamide (MEGA-10); cholic acid; sodium cholate; deoxycholic acid; sodium deoxycholate; taurocholic acid; sodium taurocholate; taurodeoxycholic acid; sodium taurodeoxycholate; 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate (CHAPS); 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propane-sulfonate (CHAPSO); -dodecyl-N,N-dimethyl-3-ammonio-1-propane-sulfonate (ZWITTERGENT 3-12); N,N-bis-(3-D-gluconeamidopropyl)-deoxycholamide (DEOXY-BIGCHAP); -octylglucoside; sucrose monolaurate; glycocholic acid/sodium glycocholate; laurosarcosine (sodium salt); glycodeoxycholic acid/sodium glycodeoxycholate; sodium dodceyl sulfate (SDS); 3-(trimethylsilyl)-1-propanesulfonic acid (DSS); cetrimide (CTAB, the principal component of which is hexadecyltrimethylammonium bromide); hexadecyltrimethylammonium bromide; dodecyltrimethylammonium bromide; hexadecyltrimethyl-ammonium bromide; tetradecyltrimethylammonium bromide; benzyl dimethyldodecylammonium bromide; benzyl dimethyl-hexadecylammonium chloride; and benzyl dimethyltetra-decylammonium bromide. The above detergents are commercially available from e.g., Sigma Chemical Co., St. Louis, Mo. Various cationic lipids known in the art can also be used as detergents. See Balasubramaniam et al., 1996, Gene Ther., 3:163-72 and Gao, X., and L. Huang. 1995, Gene Ther., 2:7110-722.
A wide variety of other methods can be used to deliver the expression constructs to cells. Such methods include DEAE dextran-mediated transfection, calcium phosphate precipitation, polylysine- or polyornithine-mediated transfection, or precipitation using other insoluble inorganic salts, such as strontium phosphate, aluminum silicates including bentonite and kaolin, chromic oxide, magnesium silicate, talc, and the like. Other useful methods of transfection include electroporation, sonoporation, protoplast fusion, liposomes, peptoid delivery, or microinjection. See, e.g., Sambrook et al., supra, for a discussion of techniques for transforming cells of interest; and Felgner, P. L., Advanced Drug Delivery Reviews (1990) 5:163-187, for a review of delivery systems useful for gene transfer. Methods of delivering DNA using electroporation are described in, e.g., U.S. Pat. Nos. 6,132,419; 6,451,002, 6,418,341, 6,233,483, U.S. Patent Publication No. 2002/0146831; and International Publication No. WO/0045823, all of which are incorporated herein by reference in their entireties.
Moreover, the HCV polynucleotides can be adsorbed to, or entrapped within, an ISCOM. Classic ISCOMs are formed by combination of cholesterol, saponin, phospholipid, and immunogens, such as viral envelope proteins. Generally, the HCV molecules (usually with a hydrophobic region) are solubilized in detergent and added to the reaction mixture, whereby ISCOMs are formed with the HCV molecule incorporated therein. ISCOM matrix compositions are formed identically, but without viral proteins. Proteins with high positive charge may be electrostatically bound in the ISCOM particles, rather than through hydrophobic forces. For a more detailed general discussion of saponins and ISCOMs, and methods of formulating ISCOMs, see Barr et al. (1998) Adv. Drug Delivery Reviews 32:247-271 (1998); U.S. Pat. Nos. 4,981,684, 5,178,860, 5,679,354 and 6,027,732, incorporated herein by reference in their entireties; European Publ. Nos. EPA 109,942; 180,564 and 231,039; and Coulter et al. (1998) Vaccine 16:1243.
Additionally, biolistic delivery systems employing particulate carriers such as gold and tungsten, are especially useful for delivering the expression constructs of the present invention. The particles are coated with the construct to be delivered and accelerated to high velocity, generally under a reduced atmosphere, using a gun powder discharge from a “gene gun.” For a description of such techniques, and apparatuses useful therefore, see, e.g., U.S. Pat. Nos. 4,945,050; 5,036,006; 5,100,792; 5,179,022; 5,371,015; and 5,478,744.
Compositions Comprising Fusion Proteins or PolynucleotidesThe invention also provides compositions comprising the fusion proteins or polynucleotides, as well as compositions including the individual components of these fusion proteins or polynucleotides. Compositions of the invention preferably comprise a pharmaceutically acceptable carrier. The carrier should not itself induce the production of antibodies harmful to the host. Pharmaceutically acceptable carriers are well known to those in the art. Such carriers include, but are not limited to, large, slowly metabolized, macromolecules, such as proteins, polysaccharides such as latex functionalized sepharose, agarose, cellulose, cellulose beads and the like, polylactic acids, polyglycolic acids, polymeric amino acids such as polyglutamic acid, polylysine, and the like, amino acid copolymers, and inactive virus particles.
Pharmaceutically acceptable salts can also be used in compositions of the invention, for example, mineral salts such as hydrochlorides, hydrobromides, phosphates, or sulfates, as well as salts of organic acids such as acetates, proprionates, malonates, or benzoates. Especially useful protein substrates are serum albumins, keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, tetanus toxoid, and other proteins well known to those of skill in the art. Compositions of the invention can also contain liquids or excipients, such as water, saline, glycerol, dextrose, ethanol, or the like, singly or in combination, as well as substances such as wetting agents, emulsifying agents, or pH buffering agents. Liposomes can also be used as a carrier for a composition of the invention, such liposomes are described above.
If desired, co-stimulatory molecules which improve immunogen presentation to lymphocytes, such as B7-1 or B7-2, or cytokines such as GM-CSF, IL-2, and IL-12, can be included in a composition of the invention. Optionally, adjuvants can also be included in a composition. Adjuvants which can be used include, but are not limited to: (1) aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc.; (2) oil-in-water emulsion formulations (with or without other specific immunostimulating agents such as muramyl peptides (see below) or bacterial cell wall components), such as for example (a) MF59 (U.S. Pat. No. 6,299,884, incorporated herein by reference in its entirety; Chapter 10 in Vaccine design: the subunit and adjuvant approach, eds. Powell & Newman, Plenum Press 1995), containing 5% Squalene, 0.5% TWEEN 80™, and 0.5% SPAN 85™ (optionally containing various amounts of MTP-PE (see below), although not required) formulated into submicron particles using a microfluidizer such as Model 110Y microfluidizer (Microfluidics, Newton, Mass.), (b) SAF, containing 10% Squalane, 0.4% TWEEN 80™, 5% pluronic-blocked polymer L121, and thr-MDP either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion, and (c) RIBI™ adjuvant system (RAS), (Ribi Immunochem, Hamilton, Mont.) containing 2% Squalene, 0.2% TWEEN 80™, and one or more bacterial cell wall components from the group consisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL+CWS (DETOX™); (3) saponin adjuvants, such as QS21 or STIMULON™ (Cambridge Bioscience, Worcester, Mass.) may be used or particles generated therefrom such as ISCOMs (immunostimulating complexes), which ISCOMs may be devoid of additional detergent, see, e.g., International Publication No. WO 00/07621; (4) Complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA); (5) cytokines, such as interleukins (IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12 (International Publication No. WO 99/44636), etc.), interferons (e.g., gamma interferon), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), etc.; (6) detoxified mutants of a bacterial ADP-ribosylating toxin such as a cholera toxin (CT), a pertussis toxin (PT), or an E. coli heat-labile toxin (LT), particularly LT-K63 (where lysine is substituted for the wild-type amino acid at position 63) LT-R72 (where arginine is substituted for the wild-type amino acid at position 72), CT-S109 (where serine is substituted for the wild-type amino acid at position 109), and PT-K9/G129 (where lysine is substituted for the wild-type amino acid at position 9 and glycine substituted at position 129) (see, e.g., International Publication Nos. W093/13202 and W092/19265); (7) MPL or 3-O-deacylated MPL (3dMPL) (see, e.g., GB 2220221), EP-A-0689454, optionally in the substantial absence of alum when used with pneumococcal saccharides (see, e.g., International Publication No. WO 00/56358); (8) combinations of 3dMPL with, for example, QS21 and/or oil-in-water emulsions (see, e.g., EP-A-0835318, EP-A-0735898, EP-A-0761231; (9) oligonucleotides comprising CpG motifs (see, e.g., Roman et al. (1997) Nat. Med. 3:849-854; Weiner et al. (1997) Proc. Natl. Acad. Sci. USA 94:10833-10837; Davis et al. (1998) J. Immunol. 160:870-876; Chu et al. (1997) J. Exp. Med. 186:1623-1631; Lipford et al. (1997) Eur. J. Immunol. 27:2340-2344; Moldoveanu et al. (1988) Vaccine 16:1216-1224; Krieg et al. (1995) Nature 374:546-549; Klinman et al. (1996) Proc. Natl. Acad. Sci. USA 93:2879-2883; Ballas et al. (1996) J. Immunol. 157:1840-1845; Cowdery et al. (1996) J. Immunol. 156:4570-4575; Halpern et al. (1996) Cell Immunol. 167:72-78; Yamamoto et al. (1988) Jpn. J. Cancer Res. 79:866-873; Stacey et al. (1996) J. Immunol. 157:2116-2122; Messina et al. (1991) J. Immunol. 147:1759-1764; Yi et al. (1996) J. Immunol. 157:4918-4925; Yi et al. (1996) J. Immunol. 157:5394-5402; Yi et al. (1998) J. Immunol. 160:4755-4761; Yi et al. (1998) J. Immunol. 160:5898-5906; International Publication Nos. WO 96/02555, WO 98/16247, WO 98/18810, WO 98/40100, WO 98/55495, WO 98/37919 and WO 98/52581), such as those containing at least on CG dinucleotide, with cytosine optionally replaced with 5-methylcytosine; (10) a polyoxyethylene ether or a polyoxyethylene ester (see, e.g., International Publication No. WO 99/52549); (11) a polyoxyethylene sorbitan ester surfactant in combination with an octoxynol (see, e.g., International Publication No. WO 01/21207) or a polyoxyethylene alkyl ether or ester surfactant in combination with at least one additional non-ionic surfactant such as an octoxynol (see, e.g., International Publication No. WO 01/21152); (12) a saponin and an immunostimulatory oligonucleotide such as a CpG oligonucleotide (see, e.g., International Publication No. WO 00/62800); (13) an immunostimulant and a particle of metal salt (see, e.g., International Publication No. WO 00/23105); and (14) other substances that act as immunostimulating agents to enhance the effectiveness of the composition.
Muramyl peptides include, but are not limited to, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acteyl-normuramyl-L-alanyl-D-isogluatme (nor-MDP), -acetylmuramyl-L-alanyl-D-isogluatminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-huydroxyphosphoryloxy)-ethylamine (MTP-PE), etc.
Moreover, the HCV proteins can be adsorbed to, or entrapped within, an ISCOM, as described above. Additionally, ISCOMs with adsorbed HCV core proteins, either the entire core region or a fragment of HCV core protein, may be added to the formulations. Most preferably, the HCV core protein is a fragment comprising a polypeptide from the region spanning amino acid positions 121-135. See, e.g., International Publication No. WO 01/37869A, incorporated herein by reference in its entirety.
As explained above, the composition may also contain immunostimulatory molecules, either in addition to or in place of the antigen delivery system. Immunostimulatory agents for use herein include, without limitation, monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL+CWS (Detox™). MPL may be formulated into an emulsion to enhance its immunostimulatory affect. See, e.g., Ulrich et al., “MPLr immunostimulat: adjuvant formulations.” in Vaccine Adjuvants: Prepartion Methods and Research Protocols (O'Hagan D T, ed.) Human Press Inc., NJ (2000) pp. 273-282. MPL has been shown to induce the synthesis and release of cytokines, particularly IL-2 and IFN-γ. Other useful immunostimulatory molecules include LPS and immunostimulatory nucleic acid sequences (ISS), including but not limited to, unmethylated CpG motifs, such as CpG oligonucleotides.
Oligonucleotides containing unmethylated CpG motifs have been shown to induce activation of B cells, NK cells and antigen-presenting cells (APCs), such as monocytes and macrophages. See, e.g., U.S. Pat. No. 6,207,646. Thus, adjuvants derived from the CpG family of molecules, CpG dinucleotides and synthetic oligonucleotides which comprise CpG motifs (see, e.g., Krieg et al. Nature (1995) 374:546 and Davis et al. J. Immunol. (1998) 160:870-876) such as any of the various immunostimulatory CpG oligonucleotides disclosed in U.S. Pat. No. 6,207,646, may be used in the subject methods and compositions. Such CpG oligonucleotides generally comprise at least 8 up to about 100 basepairs, preferably 8 to 40 basepairs, more preferably 15-35 basepairs, preferably 15-25 basepairs, and any number of basepairs between these values. For example, oligonucleotides comprising the consensus CpG motif, represented by the formula 5′-X1CGX2-3′, where X1 and X2 are nucleotides and C is unmethylated, will find use as immunostimulatory CpG molecules. Generally, X1 is A, G or T, and X2 is C or T. Other useful CpG molecules include those captured by the formula 5′-X1X2CGX3X4, where X1 and X2 are a sequence such as GpT, GpG, GpA, ApA, ApT, ApG, CpT, CpA, CpG, TpA, TpT or TpG, and X3 and X4 are TpT, CpT, ApT, ApG, CpG, TpC, ApC, CpC, TpA, ApA, GpT, CpA, or TpG, wherein “p” signifies a phosphate bond. Preferably, the oligonucleotides do not include a GCG sequence at or near the 5′- and/or 3′ terminus. Additionally, the CpG is preferably flanked on its 5′-end with two purines (preferably a GpA dinucleotide) or with a purine and a pyrimidine (preferably, GpT), and flanked on its 3′-end with two pyrimidines, preferably a TpT or TpC dinucleotide. Thus, preferred molecules will comprise the sequence GACGTT, GACGTC, GTCGTT or GTCGCT, and these sequences will be flanked by several additional nucleotides. The nucleotides outside of this central core area appear to be extremely amendable to change.
Moreover, the CpG oligonucleotides for use herein may be double- or single-stranded. Double-stranded molecules are more stable in vivo while single-stranded molecules display enhanced immune activity. Additionally, the phosphate backbone may be modified, such as phosphorodithioate-modified, in order to enhance the immunostimulatory activity of the CpG molecule. As described in U.S. Pat. No. 6,207,646, CpG molecules with phosphorothioate backbones preferentially activate B-cells, while those having phosphodiester backbones preferentially activate monocytic (macrophages, dendritic cells and monocytes) and NK cells.
One exemplary CpG oligonucleotide for use in the present compositions has the sequence 5′-TCCATGACGTTCCTGACGTT-3′ (SEQ ID NO:6).
CpG molecules can readily be tested for their ability to stimulate an immune response using standard techniques, well known in the art. For example, the ability of the molecule to stimulate a humoral and/or cellular immune response is readily determined using the immunoassays described above. Moreover, the antigen and adjuvant compositions can be administered with and without the CpG molecule to determine whether an immune response is enhanced.
The HCV proteins may also be encapsulated, adsorbed to, or associated with, particulate carriers, as described above with reference to the HCV polynucleotides. As explained above, examples of particulate carriers include those derived from polymethyl methacrylate polymers, as well as microparticles derived from poly(lactides) and poly(lactide-co-glycolides), known as PLG. See, e.g., Jeffery et al., Pharm. Res. (1993) 10:362-368; and McGee et al., J. Microencap. (1996). One preferred method for adsorbing macromolecules onto prepared microparticles is described above and in International Publication No. WO 00/050006, incorporated herein by reference in its entirety.
Methods of Producing HCV-Specific AntibodiesThe HCV proteins can be used to produce HCV-specific polyclonal and monoclonal antibodies. HCV-specific polyclonal and monoclonal antibodies specifically bind to HCV antigens. Polyclonal antibodies can be produced by administering the fusion protein to a mammal, such as a mouse, a rabbit, a goat, or a horse. Serum from the immunized animal is collected and the antibodies are purified from the plasma by, for example, precipitation with ammonium sulfate, followed by chromatography, preferably affinity chromatography. Techniques for producing and processing polyclonal antisera are known in the art.
Monoclonal antibodies directed against HCV-specific epitopes present in the proteins can also be readily produced. Normal B cells from a mammal, such as a mouse, immunized with an HCV protein, can be fused with, for example, HAT-sensitive mouse myeloma cells to produce hybridomas. Hybridomas producing HCV-specific antibodies can be identified using RIA or ELISA and isolated by cloning in semi-solid agar or by limiting dilution. Clones producing HCV-specific antibodies are isolated by another round of screening.
Antibodies, either monoclonal and polyclonal, which are directed against HCV epitopes, are particularly useful for detecting the presence of HCV or HCV antigens in a sample, such as a serum sample from an HCV-infected human. An immunoassay for an HCV antigen may utilize one antibody or several antibodies. An immunoassay for an HCV antigen may use, for example, a monoclonal antibody directed towards an HCV epitope, a combination of monoclonal antibodies directed towards epitopes of one HCV polypeptide, monoclonal antibodies directed towards epitopes of different HCV polypeptides, polyclonal antibodies directed towards the same HCV antigen, polyclonal antibodies directed towards different HCV antigens, or a combination of monoclonal and polyclonal antibodies. Immunoassay protocols may be based, for example, upon competition, direct reaction, or sandwich type assays using, for example, labeled antibody. The labels may be, for example, fluorescent, chemiluminescent, or radioactive.
The polyclonal or monoclonal antibodies may further be used to isolate HCV particles or antigens by immunoaffinity columns. The antibodies can be affixed to a solid support by, for example, adsorption or by covalent linkage so that the antibodies retain their immunoselective activity. Optionally, spacer groups may be included so that the antigen binding site of the antibody remains accessible. The immobilized antibodies can then be used to bind HCV particles or antigens from a biological sample, such as blood or plasma. The bound HCV particles or antigens are recovered from the column matrix by, for example, a change in pH.
HCV-Specific T CellsHCV-specific T cells that are activated by the above-described fusions and E1E2 complexes, including the NS3NS4NS5a fusion protein or NS3NS4NS5aNS5b fusion protein, and the E1E2 complexes, expressed in vivo or in vitro, preferably recognize an epitope of an HCV polypeptide such as an E1, E2, NS3, NS4, NS5a, NS5b polypeptide, including an epitope of an NS3NS4NS5a fusion protein or an NS3NS4NS5aNS5b fusion protein, or an E1E2 complex. HCV-specific T cells can be CD8+ or CD4+.
HCV-specific CD8+ T cells preferably are cytotoxic T lymphocytes (CTL) which can kill HCV-infected cells that display E1, E2, NS3, NS4, NS5a, NS5b epitopes complexed with an MHC class I molecule. HCV-specific CD8+ T cells may also express interferon-γ (IFN-γ). HCV-specific CD8+ T cells can be detected by, for example, 51Cr release assays (see the examples). 51Cr release assays measure the ability of HCV-specific CD8+ T cells to lyse target cells displaying an E1, E2, E1E2, NS3, NS4, NS5a, NS5b, NS3NS4NS5a, or NS3NS4NS5aNS5b epitope. HCV-specific CD8+ T cells which express IFN-γ can also be detected by immunological methods, preferably by intracellular staining for IFN-γ after in vitro stimulation with an E1, E2, NS3, an NS4, an NS5a, or an NS5b polypeptide (see the examples).
HCV-specific CD4+ cells activated by the above-described E1E2 complexes and fusions, such as an E1 polypeptide, an E2 polypeptide, an E1E2 complex, NS3NS4NS5a or NS3NS4NS5aNS5b fusion protein, expressed in vivo or in vitro, preferably recognize an epitope of an E1, E2, NS3, NS4, NS5a, or NS5b polypeptide, including an epitope of an E1E2 complex, NS3NS4NS5a or NS3NS4NS5aNS5b fusion protein, that is bound to an MHC class II molecule on an HCV-infected cell and proliferate in response to stimulating E1E2 complexes with NS3NS4NS5a or NS3NS4NS5aNS5b peptides, with or without a core polypeptide.
HCV-specific CD4+ T cells can be detected by a lymphoproliferation assay (see examples). Lymphoproliferation assays measure the ability of HCV-specific CD4+ T cells to proliferate in response to an E1, E2, NS3, an NS4, an NS5a, or an NS5b epitope.
Methods of Activating HCV-Specific T Cells.The HCV proteins or polynucleotides can be used to stimulate an immune response, such as to activate HCV-specific T cells either in vitro or in vivo. Activation of HCV-specific T cells can be used, inter alia, to provide model systems to optimize CTL responses to HCV and to provide prophylactic or therapeutic treatment against HCV infection. For in vitro activation, proteins are preferably supplied to T cells via a plasmid or a viral vector, such as an adenovirus vector, as described above.
Polyclonal populations of T cells can be derived from the blood, and preferably from peripheral lymphoid organs, such as lymph nodes, spleen, or thymus, of mammals that have been infected with an HCV. Preferred mammals include mice, chimpanzees, baboons, and humans. The HCV serves to expand the number of activated HCV-specific T cells in the mammal. The HCV-specific T cells derived from the mammal can then be restimulated in vitro by adding, e.g., HCV E1E2 and NS3NS4NS5a or NS3NS4NS5aNS5b epitopic peptides, with or without a core polypeptide, to the T cells. The HCV-specific T cells can then be tested for, inter alia, proliferation, the production of IFN-γ, and the ability to lyse target cells displaying E1E2, NS3NS4NS5a or NS3NS4NS5aNS5b epitopes in vitro.
In a lymphoproliferation assay (see examples), HCV-activated CD4+ T cells proliferate when cultured with an NS3, NS4, NS5a, NS5b, NS3NS4NS5a, or NS3NS4NS5aNS5b epitopic peptide, but not in the absence of an epitopic peptide. Thus, particular E1, E2, NS3, NS4, NS5a, NS5b, NS3NS4NS5a and NS3NS4NS5aNS5b epitopes that are recognized by HCV-specific CD4+ T cells can be identified using a lymphoproliferation assay.
Similarly, detection of IFN-γ in HCV-specific CD8+ T cells after in vitro stimulation with the above-described HCV proteins, can be used to identify E1, E2, E1E2, NS3, NS4, NS5a, NS5b, NS3NS4NS5a, and NS3NS4NS5aNS5b epitopes that particularly effective at stimulating CD8+ T cells to produce IFN-γ (see examples).
Further, 51Cr release assays are useful for determining the level of CTL response to HCV. See Cooper et al. Immunity 10:439-449. For example, HCV-specific CD8+ T cells can be derived from the liver of an HCV infected mammal. These T cells can be tested in 51Cr release assays against target cells displaying, e.g., E1E2, NS3NS4NS5a and/or NS3NS4NS5aNS5b epitopes. Several target cell populations expressing different E1E2, NS3NS4NS5a and/or NS3NS4NS5aNS5b epitopes can be constructed so that each target cell population displays different epitopes of E1E2, NS3NS4NS5a and/or NS3NS4NS5aNS5b. The HCV-specific CD8+ cells can be assayed against each of these target cell populations. The results of the 51Cr release assays can be used to determine which epitopes of E1E2, NS3NS4NS5a and/or NS3NS4NS5aNS5b are responsible for the strongest CTL response to HCV. E1E2 complexes, NS3NS4NS5a fusion proteins or NS3NS4NS5aNS5b fusion proteins, with or without core polypeptides, which contain the epitopes responsible for the strongest CTL response can then be constructed using the information derived from the 51Cr release assays.
HCV proteins as described above, or polynucleotides encoding such proteins, can be administered to a mammal, such as a mouse, baboon, chimpanzee, or human, to stimulate an immune response, such as to activate HCV-specific T cells in vivo. Administration can be by any means known in the art, including parenteral, intranasal, intramuscular or subcutaneous injection, including injection using a biological ballistic gun (“gene gun”), as discussed above.
Preferably, injection of an HCV polynucleotide is used to activate T cells. In addition to the practical advantages of simplicity of construction and modification, injection of the polynucleotides results in the synthesis of a fusion protein in the host. Thus, these immunogens are presented to the host immune system with native post-translational modifications, structure, and conformation. The polynucleotides are preferably injected intramuscularly to a large mammal, such as a human, at a dose of 0.5, 0.75, 1.0, 1.5, 2.0, 2.5, 5 or 10 mg/kg.
A composition of the invention comprising the HCV proteins or polynucleotides is administered in a manner compatible with the particular composition used and in an amount which is effective to stimulate an immune response, such as to activate HCV-specific T cells as measured by, inter alia, a 51Cr release assay, a lymphoproliferation assay, or by intracellular staining for IFN-γ. The proteins and/or polynucleotides can be administered either to a mammal which is not infected with an HCV or can be administered to an HCV-infected mammal. The particular dosages of the polynucleotides or proteins in a composition will depend on many factors including, but not limited to the species, age, and general condition of the mammal to which the composition is administered, and the mode of administration of the composition. An effective amount of the composition of the invention can be readily determined using only routine experimentation. In vitro and in vivo models described above can be employed to identify appropriate doses. The amount of polynucleotide used in the example described below provides general guidance which can be used to optimize the activation of HCV-specific T cells either in vivo or in vitro. Generally, 0.5, 0.75, 1.0, 1.5, 2.0, 2.5, 5 or 10 mg of an HCV fusion and E1 and E2 polypeptides, such as an E1E2 complex, an NS3NS4NS5a or NS3NS4NS5aNS5b fusion protein or polynucleotide, with or without a core polypeptide, will be administered to a large mammal, such as a baboon, chimpanzee, or human. If desired, co-stimulatory molecules or adjuvants can also be provided before, after, or together with the compositions.
Immune responses of the mammal generated by the delivery of a composition of the invention, including activation of HCV-specific T cells, can be enhanced by varying the dosage, route of administration, or boosting regimens. Compositions of the invention may be given in a single dose schedule, or preferably in a multiple dose schedule in which a primary course of vaccination includes 1-10 separate doses, followed by other doses given at subsequent time intervals required to maintain and/or reenforce an immune response, for example, at 1-4 months for a second dose, and if needed, a subsequent dose or doses after several months.
Deposits of Strains Useful in Practicing the InventionA deposit of biologically pure cultures of the following strains was made with the American Type Culture Collection, 10801 University Boulevard, Manassas, Va. The accession number indicated was assigned after successful viability testing, and the requisite fees were paid. made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure and the Regulations thereunder (Budapest Treaty). This assures maintenance of viable cultures for a period of thirty (30) years from the date of deposit. The organisms will be made available by the ATCC under the terms of the Budapest Treaty, which assures permanent and unrestricted availability of the progeny to one determined by the U.S. Commissioner of Patents and Trademarks to be entitled thereto according to 35 U.S.C. §122 and the Commissioner's rules pursuant thereto (including 37 C.F.R. §1.12 with particular reference to 886 OG 638). Upon the granting of a patent, all restrictions on the availability to the public of the deposited cultures will be irrevocably removed.
These deposits are provided merely as convenience to those of skill in the art, and are not an admission that a deposit is required under 35 U.S.C. §112. The nucleic acid sequences of these genes, as well as the amino acid sequences of the molecules encoded thereby, are incorporated herein by reference and are controlling in the event of any conflict with the description herein. A license may be required to make, use, or sell the deposited materials, and no such license is hereby granted.
Below are examples of specific embodiments for carrying out the present invention. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. Those of skill in the art will readily appreciate that the invention may be practiced in a variety of ways given the teaching of this disclosure.
Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.
Example 1 Production of NS3NS4NS5a PolynucleotidesA polynucleotide encoding NS3NS4NS5a (approximately amino acids 1027 to 2399, numbered relative to HCV-1) (also termed “NS345a” herein) or NS5a (approximately amino acids 1973 to 2399, numbered relative to HCV-1) was isolated from an HCV. Polynucleotides encoding a methionine residue were ligated to the 5′ end of these polynucleotides and the polynucleotides were cloned into plasmid, vaccinia virus, and adenovirus vectors.
Immunization Protocols. In one immunization protocol, mice were immunized with 50 μg of plasmid DNA encoding either NS5a or encoding an NS3NS4NS5a fusion protein by intramuscular injection into the tibialis anterior. A booster injection of 107 pfu of vaccinia virus (VV)-NS5a (intraperitoneal) or 50 μg of plasmid control (intramuscular) was provided 6 weeks later.
In another immunization protocol, mice were injected intramuscularly in the tibialis anterior with 1010 adenovirus particles encoding an NS3NS4NS5a fusion protein. An intraperitoneal booster injection of 107 pfu of VV-NS5a or an intramuscular booster injection of 1010 adenovirus particles encoding NS3NS4NS5a was provided 6 weeks later.
Example 2 Immunization with DNA Encoding an NS3NS4NS5a Fusion Protein Activates HCV-Specific CD8+ T Cells51Cr Release Assay. A 51Cr release assay was used to measure the ability of HCV-specific T cells to lyse target cells displaying an NS5a epitope. Spleen cells were pooled from the immunized animals. These cells were restimulated in vitro for 6 days with the CTL epitopic peptide p214K9 (2152-HEYPVGSQL-2160; SEQ ID NO:1) from HCV-NS5a in the presence of IL-2. The spleen cells were then assayed for cytotoxic activity in a standard 51Cr release assay against peptide-sensitized target cells (L929) expressing class I, but not class II MHC molecules, as described in Weiss (1980) J. Biol. Chem. 255:9912-9917. Ratios of effector (T cells) to target (B cells) of 60:1, 20:1, and 7:1 were tested. Percent specific lysis was calculated for each effector to target ratio.
The results of the assays are shown in Tables 1 and 2. Table 1 demonstrates that immunization with plasmid DNA encoding an NS3NS4NS5a fusion protein activates CD8+ T cells which recognize and lyse target cells displaying an NS5a epitope. Surprisingly the NS5a polypeptide of the NS3NS4NS5a fusion protein was able to activate T cells even though the NS5a polypeptide was present in a fusion protein.
Similarly, Table 2 demonstrates that delivery of the NS3NS4NS5a fusion protein to mice by means of an adenovirus vector also activates CD8+ T cells which recognize and lyse target cells displaying an HCV NS5a epitope. Thus, immunization with either “naked” (plasmid) DNA encoding an NS3NS4NS5a fusion protein or adenovirus vector-encoded fusion protein can be used to activate HCV-specific T cells.
Example 3 Immunization with DNA Encoding an NS3NS4NS5a Fusion Protein Activates HCV-Specific CD8+ T-Cells which Express IFN-γIntracellular Staining for Interferon-gamma (IFN-γ). Intracellular staining for IFN-γ was used to identify the CD8+ T cells that secrete IFN-γ after in vitro stimulation with the NS5a epitope p214K9. Spleen cells of individual immunized mice were restimulated in vitro either with p214K9 or with a non-specific peptide for 6-12 hours in the presence of IL-2 and monensin. The cells were then stained for surface CD8 and for intracellular IFN-γ and analyzed by flow cytometry. The percent of CD8+ T cells which were also positive for IFN-γ was then calculated. The results of these assays are shown in Tables 1 and 2. Table 1 demonstrates that CD8+ T cells activated in response to immunization with plasmid DNA encoding an NS3NS4NS5a fusion protein also express IFN-γ. Immunization with an NS3NS4NS5a fusion protein encoded in an adenovirus also results in CD8+ HCV-specific T cells which express IFN-γ, although to a lesser extent than immunization with a plasmid-encoded NS3NS4NS5a fusion protein (Table 2).
Lymphoproliferation assay. Spleen cells from pooled immunized mice were depleted of CD8+ T cells using magnetic beads and were cultured in triplicate with either p222D, an NS5a-epitopic peptide from HCV-NS5a (2224-AELIEANLLWRQEMG-2238; SEQ ID NO:2), or in medium alone. After 72 hours, cells were pulsed with 1μ Ci per well of 3H-thymidine and harvested 6-8 hours later. Incorporation of radioactivity was measured after harvesting. The mean cpm was calculated.
As shown in Table 3, immunization with a plasmid-encoded NS3NS4NS5a fusion protein stimulates proliferation of CD4+ HCV-specific T cells. Immunization with an adenovirus vector encoding the fusion protein also resulted in stimulated proliferation of CD4+ HCV-specific T cells (Table 4).
Mice were immunized with either 10-100 μg of plasmid DNA encoding NS345a fusion protein as described in Example 1, with PLG-linked DNA encoding NS345a, described below, or with DNA encoding NS345a, delivered via electroporation (see, e.g., U.S. Pat. Nos. 6,132,419; 6,451,002, 6,418,341, 6,233,483, U.S. Patent Publication No. 2002/0146831; and International Publication No. WO/0045823, all of which are incorporated herein by reference in their entireties, for this delivery technique). The immunizations were followed by a booster injection 6 weeks later of 1×107 pfu vaccinia virus encoding NS5a, plasmid DNA encoding NS345a or plasmid DNA encoding NS5a each as described in Example 1.
PLG-delivered DNA. The polylactide-co-glycolide (PLG) polymers were obtained from Boehringer Ingelheim, U.S.A. The PLG polymer used in this study was RG505, which has a copolymer ratio of 50/50 and a molecular weight of 65 kDa (manufacturers data). Cationic microparticles with adsorbed DNA were prepared using a modified solvent evaporation process, essentially as described in Singh et al., Proc. Natl. Acad. Sci. USA (2000) 97:811-816. Briefly, the microparticles were prepared by emulsifying 10 ml of a 5% w/v polymer solution in methylene chloride with 1 ml of PBS at high speed using an IKA homogenizer. The primary emulsion was then added to 50 ml of distilled water containing cetyl trimethyl ammonium bromide (CTAB) (0.5% w/v). This resulted in the formation of a w/o/w emulsion which was stirred at 6000 rpm for 12 hours at room temperature, allowing the methylene chloride to evaporate. The resulting microparticles were washed twice in distilled water by centrifugation at 10,000 g and freeze dried. Following preparation, washing and collection, DNA was adsorbed onto the microparticles by incubating 100 mg of cationic microparticles in a 1 mg/ml solution of DNA at 4 C for 6 hours. The microparticles were then separated by centrifugation, the pellet washed with TE buffer and the microparticles were freeze dried.
CTL activity and IFN-γ expression were measured by 51Cr release assay or intracellular staining as described in examples 2 and 3 respectively. The results are shown in Table 5.
Results demonstrate that immunization using plasmid DNA encoding for NS345a to prime mice results in activation of CD8+ HCV specific T cells.
Alphavirus replicon particles, for example, SINCR (DC+) were prepared as described in Polo et al., Proc. Natl. Acad. Sci. USA (1999) 96:4598-4603. Mice were injected with 5×106 IU SINCR (DC+) replicon particles encoding for NS345a intramuscularly (IM) as described in Example 1, or subcutaneously (S/C) at the base of the tail (BoT) and foot pad (FP), or with a combination of ⅔ of the DNA delivered via IM administration and ⅓ via a BoT route. The immunizations were followed by a booster injection of vaccinia virus encoding NS5a as described in Example 1.
IPN-γ expression was measured by intracellular staining as described in Example 3. The results are shown in Table 6. The results demonstrate that immunization via SINCR (DC+) replicon particles encoding for NS345a by a variety of routes results in CD8+ HCV specific T cells which express IFN-γ.
Alphavirus replicon particles, for example, SINCR (DC+) and SINCR (LP) were prepared as described in Polo et al., Proc. Natl. Acad. Sci. USA (1999) 96:4598-4603. Mice were immunized with 1×103 to 1×107 IU of SINCR (DC+) or SINCR (LP) replicon particles encoding for NS345a, by intramuscular injection into the tibialis anterior, followed by a booster injection of 107 pfu vaccinia virus encoding NS5a at 6 weeks.
IFN-γ expression was measured by intracellular staining as described in Example 3. Administration of an increase in the number of SINCR (DC+) replicon particles encoding NS345a resulted in an increase in % of CD8+ T cells expressing IFN-γ.
Example 8 Alphavirus Replicon Priming, Followed by Various Boosting RegimesAlphavirus replicon particles, for example, SINCR (DC+) were prepared as described in Polo et al., Proc. Natl. Acad. Sci. USA (1999) 96:4598-4603. Mice were primed with SINCR (DC+), 1.5×106 IU replicon particles encoding NS345a, by intramuscular injection into the tibialis anterior, followed by a booster of either 10-100 μg of plasmid DNA encoding for NS5a, 1010 adenovirus particles encoding NS345a, 1.5×106 IU SINCR (DC+) replicon particles encoding NS345a, or 107 pfu vaccinia virus encoding NS5a at 6 weeks.
IFN-γ expression was measured by intracellular staining as described in Example 3. The results are shown in Table 7. The results demonstrate that boosting with vaccinia virus encoding NS5a DNA results in the strongest generation of CD8+ HCV specific T cells which express IFN-γ. Boosting with plasmid encoding NS5a DNA also results in a good response, while lesser responses are noted with adenovirus NS345a or SINCR DC+boosted animals.
Alphavirus replicon particles, for example, SINCR (DC+) and SINCR (LP) were prepared as described in Polo et al., Proc. Natl. Acad. Sci. USA (1999) 96:4598-4603. Mice were immunized with 1×102 to 1×106 IU SINCR (DC+) replicons encoding NS345a via a combination of delivery routes (⅔ IM and ⅓ S/C) as well as by S/C alone, or with 1×102 to 1×106 IU SINCR (LP) replicon particles encoding NS345a via a combination of delivery routes (⅔ IM and ⅓ S/C) as well as by S/C alone. The immunizations were followed by a booster injection of 107 pfu vaccinia virus encoding NS5a at 6 weeks.
IFN-γ expression was measured by intracellular staining as described in Example 3. The results are shown in
Mice were immunized with either 10-100 μg of plasmid DNA encoding NS5a as described in Example 1 or with PLG-linked DNA encoding NS5a as described in Example 5. The immunizations were followed by a booster injection at 6 weeks of either 10-100 μg of plasmid DNA encoding for NS5a, 1010 adenovirus particles encoding NS345a, 1.5×106 IU SINCR (DC+) replicon particles encoding NS345a, or 107 pfu vaccinia virus encoding NS5a.
CTL activity and IFN-γ expression were measured by the methods described in Examples 2 and 3.
The results are shown in Table 8. The results demonstrate that priming with plasmid DNA encoding for NS5a or PLG-linked DNA encoding NS5a results in activation of CD8+ HCV specific T cells.
Mice were immunized with 10-100 μg of plasmid DNA encoding NS34b by intramuscular injection to the tibialis anterior or with PLG linked DNA encoding NS5a as described in Example 5. The immunizations were followed by a booster injection of plasmid DNA encoding for NS5a as described in Example 1.
CTL activity and IFN-γ expression were measured by the methods described in Examples 2 and 3.
The results are shown in Table 9. The results demonstrate that priming with plasmid DNA encoding NS345b or PLG-linked NS345b results in activation of CD8+ HCV specific T cells.
Mice were immunized with 100 μg plasmid DNA encoding for NS345a or with 100 μg PLG-linked DNA encoding NS345a. Additionally, separate DNA plasmids encoding NS5a, NS34a, and NS4ab (33.3 μg each) were administered concurrently to another group of mice. Finally, PLG-linked DNA encoding NS5a, NS34a, and NS4ab (33.3 μg each) were administered concurrently to another group of mice. The immunizations were followed by a booster injection of 1×107 pfu vaccinia virus encoding NS5a, 6 weeks post first immunization.
IFN-γ expression was measured by the method described in Example 3. The results are shown in
Groups of 10 C57 black mice were immunized IM at 0, 21 and 60 days with the formulations shown in Table 10. The NS345Core121-PLGdss group received a vaccine dose of 50 μl in each leg whereas the other vaccine groups received a vaccine dose of 50 μl in one leg.
NS345Core121-ISCOMS were comprised of amino acids 1242 to 3011 and 1-121 and the HCV polyprotein, numbered relative to HCV-1 and were adsorbed to ISCOMS with a ratio of protein to QH of approximately 8:1, using standard techniques. See, e.g., International Publication No. WO 01/37869A, incorporated herein by reference in its entirety.
Core-ISCOMS including an HCV core protein fragment from the region spanning amino acid positions 1-191 of the HCV polyprotein, numbered relative to HCV-1, with a ratio of protein to QH of 1:1, were produced using standard techniques. See, e.g., International Publication No. WO 01/37869A, incorporated herein by reference in its entirety.
NS345Core121 was formulated in 0.1% SDS in PBS and contained DTT. Protein was diluted in PBS and mixed 1:1 with MF59 (see, Ott et al., “MF59—Design and Evaluation of a Safe and Potent Adjuvant for Human Vaccines” in Vaccine Design: The Subunit and Adjuvant Approach (Powell, M. F. and Newman, M. J. eds.) Plenum Press, New York (1995) pp. 277-296; and U.S. Pat. No. 6,299,884, incorporated herein by reference in its entirety) prior to immunization.
For NS345Core121-PLGdss, PLG microparticles produced as described above were treated with 3-(trimethylsilyl)-1-propanesulfonic acid (DSS) to enhance adsorption of antigen. DSS is commercially available from, e.g., Sigma Chemical Co., St. Louis, Mo. NS345Core121 was adsorbed thereto using standard techniques (see, International Publication No. WO 00/050006). The NS345Core121-PLGdss was mixed with MF59 prior to immunization.
As shown in Table 10, NS345Core121-ISCOMS produced antibody response only to NS5 in immunized C57 black mice. Higher levels of antibodies to NS5 were produced in mice immunized with NS345Core121 adjuvanted with MF59, however no antibody response to core, NS3 or NS4 was produced with this adjuvant either.
Mice immunized with Core-ISCOMS produced antibodies to core. In contrast, NS345Core121-PLGdss immunized mice produced significantly higher antibodies to NS5 than NS345Core121-ISCOMS. In addition, NS345Core121-PLGdss immunized mice produced antibodies to NS3 and some antibody response to core, but no antibodies to NS4.
Groups of 10 C57 black mice were immunized IM at 0, 30 and 60 days with the formulations shown in Tables 11 and 12. For the studies shown in Table 11, the NS345 and NS345Core121 protein concentration was 10 μg per dose, and for those in Table 12, the concentration was 5 μg per dose.
For PLG-NS345 (amino acids 1242 to 3011 of the HCV polyprotein) and PLG-NS345Core121 (amino acids 1242-3011 and 1-121 of the HCV polyprotein), PLG microparticles were prepared and NS345 or NS345Core121 were adsorbed thereto using standard techniques, as described above.
For PLG-NS345+PLG-CTAB-E1E2 DNA, PLG microparticles were prepared and NS345 was adsorbed to the microparticles as described above. E1E2 DNA was produced as follows. Mammalian expression plasmid pMH-E1E2-809 (
To produce the PLG-CTAB-E1E2 DNA, PLG microparticles were treated with CTAB as described above (see, International Publication No. WO 00/050006).
For PLG-NS345Core121+E1E2 DNA PLG-NS345Core121 and E1E2 DNA were produced as described above.
For PLG-NS345 or PLG-NS345Core121+MF59, PLG-NS345 or PLG-NS345Core121, was combined with MF59 as described above.
For PLG-NS345 or PLG-NS345Core121+CTAB-CpG, NS345 or NS345Core121 was adsorbed to PLG as described above. The CpG molecule used was 5′-TCCATGACGTTCCTGACGTT-3′ and this was treated with CTAB, as described above.
For PLG-NS345 or PLG-NS345Core121+QS21, the saponin adjuvant QS21 was combined with the PLG-HCV proteins.
For PLG-NS345 or PLG-NS345Core121+CTAB-CpG+MF59, the various components, as described above, were combined.
The remaining adjuvants used in the studies and shown in the tables are self-explanatory.
The results of these studies are shown in Tables 11 and 12. As can be seen in Table 11, none of the formulations produced antibody responses to core, NS3 or NS4 antigens. However, PLG-NS345+CTAB-CPG in MF59 produced the highest antibody titers to NS5. PLG-NS345Core121+QS21, PLG-NS345+CTAB-CPG, PLG-NS345Core121+CTAB-CPG, and PLG-NS345+QS21 produced moderate antibody titers to NS5. The other formulations produced very low antibody titers to NS5.
As can be seen in Table 12, NS345Core121/MF59/MPL and NS345Core121/MF59/CpG formulations produced very high antibody titers to NS345Core121, NS345Core121/MF59, NS345/MF59/CpG, and NS345Core121/MF59/Cho1/QS21 formulations produced moderate antibody titers to NS345Core121. The other formulations produced very low or no antibody titers to NS345Core121.
Groups of 8 C57 black mice were immunized IM at 0, 30 and 60 days with the formulations shown in Table 13 and are as described above. Spleens were obtained after the last immunization. The NS345Core121 protein concentration was 25 μg per dose. The NS34a, NS5b and core doses were 3 μg each.
The results of this study are shown in Table 13. As can be seen, NS345Core121/Alum/CpG, PLG-NS345Core121/PLG/CpG, NS34a+NS5B+Core/MF59/CpG and PLG-NS345Core121/MF59/CpG formulations demonstrated strong LPA responses to NS5, NS34 and core antigens. The NS345Core121/MF59 formulation also produced a strong LPA response to NS5 and NS34. Core was not tested. Moderate LPA responses were observed to NS5, NS34 and Core antigens with PLG-NS345Core121/MF59 and NS34a+NS5B+Core/MF59 formulations. The NS345Core121/MF59/CpG formulation may not have been administered properly in that no LPA response was observed in this experiment. In a subsequent experiment as shown in Table 14, an LPA was observed to this formulation.
Groups of 8 C57 black mice were immunized once IM with the formulations shown in Table 14, produced as described above. Draining lymph nodes were obtained. The NS345Core121 protein concentration was 25 μg per dose.
The results of this study are shown in Table 14. As can be seen in Table 14, all the formulations tested produced a strong LPA response to NS5, NS34 and Core as well as the NS345Core121.
The safety and immunogenicity of HCV proteins completed with the adjuvant, Iscomatrix, was studied in Rhesus macaques. Three groups made up of four animals each were immunized IM as detailed below at week 0, 4 and 8 weeks. Vaccines were prepared as described above. The ISCOMS used lacked QH-A.
Bleeds occurred as follows and immunogenicity was determined by CTL assays, lymphoproliferation assays, FACS analysis and antibody response a previously described (Palakos, et al. (2001) J. of Immunology 166:3589).
The immunogenicity of the different HCV recombinant protein vaccines is shown in Tables 15-17.
As can be seen in Table 15, the HCV Core-ISCOM vaccine produced no CTL positive responses in any of the 4 immunized macaques after the second or third immunizations. No positive CD8 γ-interferon and/or TNF-α intracellular staining was also observed, although backgrounds were high in these particular arrays. At least two of four macaques produced a strong LPA response after the second immunizations, but only one remained positive after the third immunization. Two of four macaques produced positive CD4 intracellular staining after the second immunization and four of four after the third immunization.
As shown in Table 16, the HCV NS345Core121-ISCOM vaccine after the second immunization produced CTL positive responses to peptide pools representing two or more HCV proteins in three of four macaques (two of these macaques had responses to peptide pools from NS3, NS5a and NS5b, one to peptide pools from core and NS3). CD8 positive γ-interferon and/or TNF-α intracellular staining to peptide pools representing two or more HCV proteins was positive in at least three of four macaques. One of four macaques produced a strong LPA response. At least three of four macaques produced CD4 positive intracellular staining to two or more HCV proteins. After the third immunization, only one of four macaques had a positive CTL response, CD8 positive intracellular staining and C04 positive intracellular staining. One other macaque had a positive LPA response and weak CD8+CD4 intracellular staining, This decline in immunogenicity was likely due to instability of the vaccine formulation (see below).
As shown in Table 17, the HCV Core-ISCOM+NS5b-ISCOM vaccine produced a CTL positive response to NS5b in one of the 4 immunized macaques after the second immunization which did not remain positive after the third immunization. CD8 positive intracellular positive staining was observed in one of four animals post second. Two of four macaques produced a strong LPA response after the second immunization which did not remain positive after the third immunization. Two other macaques did develop a strong LPA response after the third immunization. Three or four developed positive CD4 intracellular staining. One developed positive CD8 intracellular staining.
Three weeks after the third immunization, it was noted that the physical appearance of the polyprotein vaccine solution was visibly turbid. The core vaccine also was turbid but less so. The Core-NS5 vaccine was also slightly turbid. Analysis of this turbidity in the polyprotein formulation indicated that the ISCOM particles had precipitated into large aggregates. These aggregates could be dispersed by vortexing with 0.1% TWEEN 80 detergent. It is probable that this change in the formulation of the vaccine occurred before the last immunization. This observed change in appearance of the vaccines may have affected their immunogenicity as cellular immune results declined in all three vaccines.
The immunogenicity of HCV Core-ISCOMS, NS345Core121-ISCOMS and Core-ISCOMS+NS5b-ISCOMS as assessed by EIA antibody response is shown in Table 18. As can be seen, all three vaccines produced an antibody response by the third immunization to their corresponding HCV proteins, except for the NS345Core121-ISCOM vaccine. The NS345Core121-ISCOM vaccine produced antibody responses to NS3, NS4 and a very strong antibody response to NS5, but no antibody response to HCV core.
Five groups of five chimps each were immunized IM at 0, 0.7, 2 and 5 months with the formulations presented below. Blood was collected at week 0, two weeks subsequent to the second immunization, two weeks following the third immunization and two weeks after the fourth immunization.
Formulation 1: 20 μg E1E2 polypeptide+MF59+500 μg CpG (produced as described above);
Formulation 2: 1 mg NS345Core121-ISCOM (produced as described above);
Formulation 3: 6 mg each of CTAB-PLG-E1E2 (bp 574-2427, encoding amino acids 192-809 of the HCV polyprotein, numbered relative to HCV-1); CTAB-PLG-NS34a (bp 3079-5133, encoding amino acids 1027-1711 of the HCV polyprotein, numbered relative to HCV-1); CTAB-PLG-NS34ab (bp 4972-5916, encoding amino acids 1658-1972 of the HCV polyprotein, numbered relative to HCV-1); CTAB-PLG-NS5a (bp 5917-7260, encoding amino acids 1973-2420 of the HCV polyprotein, numbered relative to HCV-1);
Formulation 4: 6 mg each of E1E2 DNA, NS34a DNA, NS34ab DNA and NS5a DNA, having the same coordinates as described above, delivered without PLG via electroporation (see, e.g., U.S. Pat. Nos. 6,132,419; 6,451,002, 6,418,341, 6,233,483, U.S. Patent Publication No. 2002/0146831; and International Publication No. WO/0045823, all of which are incorporated herein by reference in their entireties, for this delivery technique). Results are shown in
As can be seen, in
As shown in
As can be seen in Tables 19 and 20, E1E2 adjuvanted with MF59 primed anti-E1E2 titers. CpG enhanced anti-E1E2 responses as well as TH1 responses and the ISCOM and the two DNA vaccines were capable of priming CD4+ and CD8+ T cell responses to HCV.
Thus, HCV polypeptides and polynucleotides, either alone or as fusions, to stimulate cell-mediated immune responses, are disclosed. Although preferred embodiments of the subject invention have been described in some detail, it is understood that obvious variations can be made without departing from the spirit and the scope of the invention as defined by the appended claims.
Claims
1. A composition comprising: wherein the composition stimulates an immune response to HCV.
- (a) a fusion protein comprising HCV polypeptides, wherein the HCV polypeptides consist of an NS3, an NS4, an NS5a, and NS5b polypeptide of a hepatitis C virus (HCV) and optionally a core polypeptide; and
- (b) a polynucleotide encoding an HCV E1E2 complex,
2. The composition of claim 1, wherein at least one of the HCV polypeptides is derived from a different strain of HCV than the other HCV polypeptides.
3. The composition of claim 1, further comprising a pharmaceutically acceptable excipient.
4. The composition of claim 3, further comprising an adjuvant.
5. The composition of claim 3, further comprising a CpG oligonucleotide.
6. The composition of claim 3, wherein said fusion protein is adsorbed to or entrapped within a microparticle or ISCOM.
7. A composition comprising a hepatitis C virus (HCV) polynucleotide encoding an HCV E1E2 complex, HCV polypeptides, and a pharmaceutically acceptable excipient, wherein the HCV polypeptides consist of: wherein the composition stimulates an immune response to HCV.
- (a) an isolated and purified NS3 polypeptide;
- (b) an isolated and purified NS4;
- (c) an isolated and purified NS5a polypeptide;
- (d) an isolated and purified NS5b polypeptide; and optionally,
- (e) an isolated and purified core polypeptide,
8. The composition of claim 7, further comprising an adjuvant.
9. The composition of claim 7, further comprising a CpG oligonucleotide.
10. The composition of claim 7, wherein one or more of said HCV polypeptides is adsorbed to or entrapped within a microparticle or ISCOM.
11. A method of activating T cells of a vertebrate subject which recognize an epitope of an HCV polypeptide, comprising the step of:
- administering the composition of claim 1 to said vertebrate subject, whereby a population of activated T cells recognizes an epitope of the NS3, NS4, NS5a, NS5b and/or core polypeptides.
12. A method of activating T cells of a vertebrate subject which recognize an epitope of an HCV polypeptide, comprising the step of:
- administering the composition of claim 4 to said vertebrate subject, whereby a population of activated T cells recognizes an epitope of the NS3, NS4, NS5a, NS5b and/or core polypeptides.
13. A method of activating T cells of a vertebrate subject which recognize an epitope of an HCV polypeptide, comprising the step of:
- administering the composition of claim 5 to said vertebrate subject, whereby a population of activated T cells recognizes an epitope of the NS3, NS4, NS5a, NS5b and/or core polypeptides.
14. A method of activating T cells of a vertebrate subject which recognize an epitope of an HCV polypeptide, comprising the step of:
- administering the composition of claim 6 to said vertebrate subject, whereby a population of activated T cells recognizes an epitope of the NS3, NS4, NS5a, NS5b and/or core polypeptides.
15. A method of activating T cells of a vertebrate subject which recognize an epitope of an HCV polypeptide, comprising the step of:
- administering the composition of claim 7 to said vertebrate subject, whereby a population of activated T cells recognizes an epitope of the NS3, NS4, NS5a, NS5b and/or core polypeptides.
Type: Application
Filed: Sep 2, 2008
Publication Date: Apr 16, 2009
Inventors: Michael Houghton (Danville, CA), Steve Coates (Emeryville, CA), Mark Selby (San Francisco, CA), Xavier Paliard (San Francisco, CA)
Application Number: 12/231,351
International Classification: A61K 39/00 (20060101);
